Methods and compositions for treating non-ERK MAPK pathway inhibitor-resistant cancers

ABSTRACT

The present invention provides, inter alia, methods, pharmaceutical compositions, and kits for treating or ameliorating the effects of a cancer in a subject, which cancer is refractory or resistant to non-ERK MAPK pathway inhibitor therapy. Also provided are methods for identifying a subject having cancer who would benefit from therapy with an ERK inhibitor and methods for inhibiting phosphorylation of RSK in a cancer cell that is refractory or resistant to a non-ERK MAPK pathway inhibitor.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit to and is a continuation application ofU.S. patent application Ser. No. 15/161,137, filed May 20, 2016. The'137 application is a continuation in part of PCT internationalapplication no. PCT/US2014/071749, filed Dec. 19, 2014, which claimsbenefit of U.S. Patent Application Ser. No. 61/919,551, filed on Dec.20, 2013 which, applications are incorporated by reference herein intheir entireties.

FIELD OF INVENTION

The present invention provides, inter alia, methods, pharmaceuticalcompositions, and kits for treating or ameliorating the effects of acancer in a subject, which cancer is refractory or resistant to non-ERKMAPK pathway inhibitor therapy.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains references to amino acids and/or nucleic acidsequences that have been filed concurrently herewith as sequence listingtext file “0375608.txt”, file size of 356 KB, created on Dec. 18, 2014.The aforementioned sequence listing is hereby incorporated by referencein its entirety pursuant to 37 C.F.R. § 1.52 (e)(5).

BACKGROUND OF THE INVENTION

Drug inhibitors that target components of the mitogen-activated proteinkinases (MAPK) signaling pathway show clinical efficacy in a variety ofcancers, particularly those bearing mutations in the BRAF proteinkinase. Both RAF and MEK inhibitors are approved for single-agent use inadvanced metastatic BRAF mutant melanoma. Either alone or incombination, BRAF and MEK inhibitor activity is unpredictable in othercancers, with promising efficacy in BRAF mutant thyroid and lung cancer,but only marginal activity in BRAF mutant colorectal cancer.

As with other targeted therapies, patterns of disease response to RAFand MEK inhibitors appear to be influenced by the intrinsic geneticheterogeneity present in the cancers where the drugs are used. Forinstance, it has been shown that certain genetic alterations, includingPTEN and other changes that activate the PI3K cell growth signalingpathway, may predict a poor initial response, and/or relatively rapidprogression, in BRAF mutant melanoma treated with the RAF inhibitorvemurafenib. Likewise, direct mutations in MEK gene loci appear toemerge in tumors that have progressed following either BRAF, MEK, orcombined drug treatment. Several additional examples, from RAS and RAFgene amplification and splicing mutations, suggest that acquired drugresistance is produced when oncogenic pleiotropy encounters theselective pressure of targeted drug treatment.

In view of the foregoing, there is a need for novel targeted agents thatwould ideally inhibit diverse nodes of oncogenic pathways, and also beeffective in combinations by inducing a burden of selective pressuresthat exceeds the adaptive capacity of diverse cancer genomes. Thepresent application is directed to meeting these and other needs.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating the effects of a cancer in a subject, which cancer isrefractory or resistant to non-ERK MAPK pathway inhibitor therapy. Themethod comprises administering to the subject an effective amount ofBVD-523 or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a method for treating orameliorating the effects of a cancer in a subject. The method comprises:

-   -   (a) identifying a subject with cancer that has become refractory        or resistant to BRAF inhibitor therapy, MEK inhibitor therapy,        or BRAF and MEK inhibitor therapy; and    -   (b) administering to the subject with said refractory or        resistant cancer an effective amount of an ERK inhibitor, which        is BVD-523 or a pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a method for treatingor ameliorating the effects of cancer in a subject, which cancer isrefractory or resistant to BRAF inhibitor therapy, MEK inhibitortherapy, or both. The method comprises administering to the subject aneffective amount of BVD-523 or a pharmaceutically acceptable saltthereof.

Another embodiment of the present invention is a method for identifyinga subject having cancer who would benefit from therapy with an ERKinhibitor. The method comprises:

(a) obtaining a biological sample from the subject; and

(b) screening the sample to determine whether the subject has one ormore of the following markers:

-   -   (i) a switch between RAF isoforms,    -   (ii) upregulation of receptor tyrosine kinase (RTK) or NRAS        signaling,    -   (iii) reactivation of mitogen activated protein kinase (MAPK)        signaling,    -   (iv) the presence of a MEK activating mutation,    -   (v) amplification of mutant BRAF,    -   (vi) STAT3 upregulation,    -   (vii) mutations in the allosteric pocket of MEK that directly        block binding of inhibitors to MEK or lead to constitutive MEK        activity,        wherein the presence of one or more of the markers confirms that        the subject's cancer is refractory or resistant to BRAF and/or        MEK inhibitor therapy and that the subject would benefit from        therapy with an ERK inhibitor, which is BVD-523 or a        pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a pharmaceuticalcomposition for treating or ameliorating the effects of a cancer in asubject, which cancer is refractory or resistant to non-ERK MAPK pathwaytherapy. The composition comprises a pharmaceutically acceptable carrieror diluent and an effective amount of BVD-523 or a pharmaceuticallyacceptable salt thereof.

Another embodiment of the present invention is a kit for treating orameliorating the effects of a cancer in a subject, which cancer isrefractory or resistant to non-ERK MAPK pathway therapy. The kitcomprises any of the pharmaceutical compositions according to thepresent invention packaged together with instructions for its use.

Another embodiment of the present invention is a method for inhibitingphosphorylation of RSK in a cancer cell that is refractory or resistantto a non-ERK MAPK pathway inhibitor. The method comprises contacting thecancer cell with an effective amount of BVD-523 or a pharmaceuticallyacceptable salt thereof for a period of time sufficient forphosphorylation of RSK in the cancer cell to be inhibited.

Another embodiment of the present invention is a method of treating asubject having an unresectable or metastatic BRAF600 mutation-positivemelanoma comprising administering to the subject 600 mg BID of BVD-523or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a composition fortreating a subject having an unresectable or metastatic BRAF600mutation-positive melanoma, the composition comprising 600 mg of BVD-523or a pharmaceutically acceptable salt thereof and optionally apharmaceutically acceptable carrier, adjuvant, or vehicle.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A-FIG. 1C show the progress of a dose escalation study in a humanmalignant melanoma cell line (A375 cells) for month 1. Varioustreatments (trametinib (a type 2 MEK inhibitor), dabrafenib (a BRAFinhibitor), and BVD-523 (an ERK1/2 inhibitor)) are as labeled.

FIG. 2A-FIG. 2H show the results of a proliferation assay that trackschanges in sensitivity to the escalated agent(s) at month 1. Varioustreatments (trametinib, dabrafenib, BVD-523, and pacitaxel) are aslabeled on the top of the graph. The caption to the right of the graphshows the various types of cells generated from the dose escalationstudy. For example, “dabrafenib” refers to the cells that have beentreated with the highest dose of dabrafenib from month 1 of the doseescalation study. Parental refers to the control cells that have notbeen treated with drugs. FIG. 2A, FIG. 2C and FIG. 2G are normalized tocontrol, whereas FIG. 2D, FIG. 2F and FIG. 2H show the raw data.

FIG. 3A-FIG. 3D show the progress of a dose escalation study in A375cells for month 2. Various treatments (trametinib, dabrafenib, andBVD-523) are as labeled.

FIG. 4A-FIG. 4H show the results of a proliferation assay that trackschanges in sensitivity to the escalated agent(s) at month 2. Varioustreatments (trametinib, dabrafenib, BVD-523, and pacitaxel) are aslabeled on the top of the graph. The caption to the right of the graphshows the various types of cells generated from the dose escalationstudy. For example, “dabrafenib” refers to the cells that have beentreated with the highest dose of dabrafenib from month 2 of the doseescalation study. Parental refers to the control cells that have notbeen treated with drugs. FIG. 4A, FIG. 4C and FIG. 4G are normalized tocontrol, whereas FIG. 4D, FIG. 4F and FIG. 4H show the raw data.

FIG. 5A-FIG. 5H show only the parental and BVD-523 cell line data fromFIG. 4A-FIG. 4H. Various treatments (trametinib, dabrafenib, BVD-523,and pacitaxel) are as labeled. FIG. 5A, FIG. 5C and FIG. 5G arenormalized to control, whereas FIG. 5D, FIG. 5F and FIG. 5H show the rawdata.

FIG. 6A-FIG. 6D show the progress of the dose escalation study in ahuman malignant cell line (A375 cells) for month 3. Various treatments(trametinib, dabrafenib, and BVD-523) are as labeled.

FIG. 7 is a histogram showing the results of a proliferation assay asapplied to cells grown in the DMSO control wells from the doseescalation assay.

FIG. 8A-FIG. 8D are a set of line graphs showing proliferation assaysfor month 3 of the study. Various treatments (trametinib, dabrafenib,BVD-523, and pacitaxel) are as labeled on the top of the graph. Thecaption to the right of the graph shows the various types of cellsgenerated from the dose escalation study. For example, “dabrafenib”refers to the cells that have been treated with the highest dose ofdabrafenib from month 3 of the dose escalation study. Parental refers tothe control cells that have not been treated with drugs.

FIG. 9A-FIG. 9D show only the parental, dabrafenib, and BVD-523 cellline data from FIG. 8A-FIG. 8D.

FIG. 10A is a dose matrix showing % inhibition of thetrametinib/dabrafenib combination in A375 cells using the Alamar Bluecell viability assay. FIG. 10B is a dose matrix showing excess overBliss for the trametinib/dabrafenib combination. FIG. 10C and FIG. 10Dshow % viability relative to DMSO only treated controls for dabrafeniband trametinib single agent treatments in A375 cells using the AlamarBlue cell viability assay. FIG. 10E shows % viability relative to DMSOonly treated controls for dabrafenib and trametinib combinationtreatments in A375 cells using the Alamar Blue cell viability assay.

FIG. 11A is a dose matrix showing % inhibition of thetrametinib/dabrafenib combination in A375 cells using the CellTiter-Glocell viability assay. FIG. 11B is a dose matrix showing excess overBliss for the trametinib/dabrafenib combination. FIG. 11C and FIG. 11Dshow % viability relative to DMSO only treated controls for dabrafeniband trametinib single agent treatments in A375 cells using theCellTiter-Glo cell viability assay. FIG. 11E shows % viability relativeto DMSO only treated controls for dabrafenib and trametinib combinationtreatments in A375 cells using the CellTiter-Glo cell viability assay.

FIG. 12A is a dose matrix showing % inhibition of the BVD-523/dabrafenibcombination in A375 cells using the Alamar Blue cell viability assay.FIG. 12B is a dose matrix showing excess over Bliss for theBVD-523/dabrafenib combination. FIG. 12C and FIG. 12D show % viabilityrelative to DMSO only treated controls for dabrafenib and BVD-523 singleagent treatments in A375 cells using the Alamar Blue cell viabilityassay. FIG. 12E shows % viability relative to DMSO only treated controlsfor dabrafenib and BVD-523 combination treatments in A375 cells usingthe Alamar Blue cell viability assay.

FIG. 13A is a dose matrix showing % inhibition of the BVD-523/dabrafenibcombination in A375 cells using the CellTiter-Glo cell viability assay.FIG. 13B is a dose matrix showing excess over Bliss for theBVD-523/dabrafenib combination. FIG. 13C and FIG. 13D show % viabilityrelative to DMSO only treated controls for dabrafenib and BVD-523 singleagent treatments in A375 cells using the CellTiter-Glo cell viabilityassay. FIG. 13E shows % viability relative to DMSO only treated controlsfor dabrafenib and BVD-523 combination treatments in A375 cells usingthe CellTiter-Glo cell viability assay.

FIG. 14A is a dose matrix showing % inhibition of the trametinib/BVD-523combination in A375 cells using the Alamar Blue cell viability assay.FIG. 14B is a dose matrix showing excess over Bliss for thetrametinib/BVD-523 combination. FIG. 14C and FIG. 14D show % viabilityrelative to DMSO only treated controls for BVD-523 and trametinib singleagent treatments in A375 cells using the Alamar Blue cell viabilityassay. FIG. 14E shows % viability relative to DMSO only treated controlsfor BVD-523 and trametinib combination treatments in A375 cells usingthe Alamar Blue cell viability assay.

FIG. 15A is a dose matrix showing % inhibition of the trametinib/BVD-523combination in A375 cells using the CellTiter-Glo cell viability assay.FIG. 15B is a dose matrix showing excess over Bliss for thetrametinib/BVD-523 combination. FIG. 15C and FIG. 15D show % viabilityrelative to DMSO only treated controls for BVD-523 and trametinib singleagent treatments in A375 cells using the CellTiter-Glo cell viabilityassay. FIG. 15E shows % viability relative to DMSO only treated controlsfor BVD-523 and trametinib combination treatments in A375 cells usingthe CellTiter-Glo cell viability assay.

FIG. 16A-FIG. 16D are a set of images showing Western blot analysis ofMAPK signaling in A375 cells after a 4 hour treatment with variousconcentrations (in nM) of BVD-523, dabrafenib (Dab), and Trametinib(Tram). 40 μg of total protein was loaded in each lane except whereindicated otherwise. In this experiment, duplicate samples werecollected. FIG. 16A and FIG. 16B show results from duplicate samples.Similarly, FIG. 16C and FIG. 16D also show results from duplicatesamples. In FIG. 16A and FIG. 16B, pRSK1 had a relatively weak signal inA375 cells compared to other markers. A different pRSK1-S380 antibodyfrom Cell Signaling (cat. #11989) was tested but did not give adetectable signal (data not shown). In FIG. 16C and FIG. 16D, pCRAF-338gave a minimal signal.

FIG. 17A-FIG. 17D are a set of images showing Western blot analysis ofMAPK signaling in a human colorectal carcinoma cell line (HCT116 cells)after a 4 hour treatment with various concentrations (in nM) of BVD-523,dabrafenib (Dab), and Trametinib (Tram). 40 μg of total protein wasloaded in each lane except where indicated otherwise. In thisexperiment, duplicate samples were collected. FIG. 17A and FIG. 17B showresults from duplicate samples. Similarly, FIG. 17C and FIG. 17D alsoshow results from duplicate samples. In FIG. 17A and FIG. 17B, pRSK1levels appear to be very low in HCT116 cells, and in FIG. 17C and FIG.17D, pCRAF-338 signal was also very weak.

FIG. 18A-FIG. 18D are a set of images showing Western blot analysis ofcell cycle and apoptosis signaling in A375 melanoma cells after a 24hour treatment with various concentrations (in nM) of BVD-523(“BVD523”), trametinib (“tram”) and/or dabrafenib (“Dab”) as labelled.50 μg of total protein was loaded in each lane except where indicatedotherwise. In this experiment, duplicate samples were collected. FIG.18A and FIG. 18B show results from duplicate samples. Similarly, FIG.18C and FIG. 18D also show results from duplicate samples. In FIG. 18Aand FIG. 18B, no band of a size corresponding to cleaved PARP (89 kDa)was apparent.

FIG. 19 shows that BVD-523 can treat acquired resistance to targeteddrugs in-vivo. A patient-derived line, ST052C, was isolated from aBRAFV600E melanoma patient that progressed following 10 months oftherapy with MAPK-pathway directed therapies. Treated ex vivo, ST052Cexhibited acquired cross-resistance to dabrafenib at 50 mg/kg BID.Meanwhile, BVD-523 was effective in ST052C as a single-agent at 100mg/kg BID.

FIG. 20 is a flowchart showing the dose escalation protocol used herein.

FIG. 21 shows a schematic of the mitogen-activated protein kinases(MAPK) pathway.

FIG. 22A-FIG. 22E show the results of single agent proliferation assays.Proliferation results are shown for treatment with BVD-523 (FIG. 22A),SCH772984 (FIG. 22B), Dabrafenib (FIG. 22C), Trametinib (FIG. 22D), andPaclitaxel (FIG. 22E).

FIG. 23A-FIG. 23O show the results of the combination of BVD-523 andDabrafenib. FIG. 23A shows a dose matrix showing inhibition (%) for thecombination in RKO parental cells. FIG. 23B-FIG. 23C show the results ofsingle agent proliferation assays for the combination in FIG. 23A. FIG.23D shows Loewe excess for the combination in FIG. 23A and FIG. 23Eshows Bliss excess for the combination in FIG. 23A. FIG. 23F shows adose matrix showing inhibition (%) for the combination in RKO MEK1(Q56P/+)-clone 1 cells. FIG. 23G-FIG. 23H show the results of singleagent proliferation assays for the combination in FIG. 23F. FIG. 23Ishows Loewe excess for the combination in FIG. 23F and FIG. 23J showsBliss excess for the combination in FIG. 23F. FIG. 23K shows a dosematrix showing inhibition (%) for the combination in RKO MEK1(Q56P/+)-clone 2 cells. FIG. 23L-FIG. 23M show the results of singleagent proliferation assays for the combination in FIG. 23K. FIG. 23Nshows Loewe excess for the combination in FIG. 23K and FIG. 23O showsBliss excess for the combination in FIG. 23K.

FIG. 24A-FIG. 24O show the results of the combination of SCH772984 andDabrafenib. FIG. 24A shows a dose matrix showing inhibition (%) for thecombination in RKO parental cells. FIG. 24B-FIG. 24C show the results ofsingle agent proliferation assays for the combination in FIG. 24A. FIG.24D shows Loewe excess for the combination in FIG. 24A and FIG. 24Eshows Bliss excess for the combination in FIG. 24A. FIG. 24F shows adose matrix showing inhibition (%) for the combination in RKO MEK1(Q56P/+)-clone 1 cells. FIG. 24G-FIG. 24H show the results of singleagent proliferation assays for the combination in FIG. 24F. FIG. 24Ishows Loewe excess for the combination in FIG. 24F and FIG. 24J showsBliss excess for the combination in FIG. 24F. FIG. 24K shows a dosematrix showing inhibition (%) for the combination in RKO MEK1(Q56P/+)-clone 2 cells. FIG. 24L-FIG. 24M show the results of singleagent proliferation assays for the combination in FIG. 24K. FIG. 24Nshows Loewe excess for the combination in FIG. 24K and FIG. 24O showsBliss excess for the combination in FIG. 24K.

FIG. 25A-FIG. 25O show the results of the combination of Trametinib andDabrafenib. FIG. 25A shows a dose matrix showing inhibition (%) for thecombination in RKO parental cells. FIG. 25B-FIG. 25C show the results ofsingle agent proliferation assays for the combination in FIG. 25A. FIG.25D shows Loewe excess for the combination in FIG. 25A and FIG. 25Eshows Bliss excess for the combination in FIG. 25A. FIG. 25F shows adose matrix showing inhibition (%) for the combination in RKO MEK1(Q56P/+)-clone 1 cells. FIG. 25G-FIG. 25H show the results of singleagent proliferation assays for the combination in FIG. 25F. FIG. 25Ishows Loewe excess for the combination in FIG. 25F and FIG. 25J showsBliss excess for the combination in FIG. 25F. FIG. 25K shows a dosematrix showing inhibition (%) for the combination in RKO MEK1(Q56P/+)-clone 2 cells. FIG. 25L-FIG. 25M show the results of singleagent proliferation assays for the combination in FIG. 25K. FIG. 25Nshows Loewe excess for the combination in FIG. 25K and FIG. 25O showsBliss excess for the combination in FIG. 25K.

FIG. 26A shows Lowe Volumes for the combinations tested. FIG. 26B showsBliss Volumes for the combinations tested. FIG. 26C shows Synergy Scoresfor the combinations tested.

FIG. 27A-FIG. 27I show the changes in MAPK and Effector PathwaySignaling in MEK acquired resistance. Isogenic RKO parental and MEK1(Q56P/+) cells were treated with compound for 4 or 24 h and thenimmuno-blotted with the indicated antibodies. Dabrafenib was the BRAFinhibitor and trametinib was the MEK inhibitor. FIG. 27A shows increasedsignaling in RKO MEK1 (Q56P/+) cells. FIG. 27B-FIG. 27C show the resultsof a 4 hour treatment in Experiment 1 (See, Example 7) in RKO Parental(27B) and RKO MEK1 (Q56P/+) (27C) cells. FIG. 27D-FIG. 27E show theresults of a 4 hour treatment in Experiment 2 (See, Example 7) in RKOParental (27D) and RKO MEK1 (Q56P/+) (27E) cells. FIG. 27F-FIG. 27G showthe results of a 4 hour treatment in Experiment 2 (See, Example 7) inRKO Parental (27F) and RKO MEK1 (Q56P/+) (27G) cells. FIG. 27H-FIG. 27Ishow a summary of results in RKO Parental (27H) and RKO MEK1 (Q56P/+)(27I) cells.

FIG. 28A-FIG. 28E show the results of the combination of BVD-523 andSCH772984. FIG. 28A shows a dose matrix showing inhibition (%) for thecombination in A375 cells. FIG. 28B-FIG. 28C show the results of singleagent proliferation assays for the combination in FIG. 28A. FIG. 28Dshows Loewe excess for the combination in FIG. 28A and FIG. 28E showsBliss excess for the combination in FIG. 28A.

FIG. 29A-FIG. 29F show discovery and characterization of the novelERK1/2 inhibitor BVD-523 (ulixertinib). FIG. 29A shows that BVD-523demonstrates inhibition in a reversible ATP-competitive manner. This isdemonstrated by a linear increase in IC₅₀ values for inhibition of ERK2with increasing ATP concentration as shown in FIG. 29B. FIG. 29C shows arepresentative plot of the dose-response curve and FIG. 29D shows a plotof IC₅₀ over time. FIG. 29E shows BVD-523 binding to ERK2 andphospho-ERK2 (pERK2), compared with negative control protein p38. FIG.29F shows BVD-523 binding to ERK2 compared with the ERK inhibitorsSCH772984 and pyrazolylpyrrole.

FIG. 30A-FIG. 30D show that BVD 523 inhibits cellular proliferation andenhances caspase 3 and caspase 7 activity in vitro. FIG. 30A shows thatBVD-523 demonstrates preferential activity in cells with MAPK pathwaymutations, as defined by the presence of mutations in RAS family membersand RAF. In addition, as shown in FIG. 30B, BVD-523 blocks sensitivecell lines in the G1 phase of the cell cycle. FIG. 30C shows thatBVD-523 induced a concentration- and time-dependent increase in caspaseactivity in the A375, WM266, and LS411N cancer cell lines after 72 hoursof exposure. FIG. 30D shows that the MAPK pathway and effector proteinsare modulated by acute (4-hour) and prolonged (24-hour) BVD-523treatment in BRAF^(V600E)-mutant A375 cells.

FIG. 31A-FIG. 31C show in vivo BVD-523 anti-tumor activity. BVD-523monotherapy inhibits tumor growth in (FIG. 31A) A375 and (FIG. 31B)Colo205 cell line xenograft models (^(a)P<0.0001, compared with vehiclecontrol; CPT-11 dosed on Day 14 and Day 18 only). Abbreviations: BID,twice daily; CMC, carboxymethylcellulose; QD, every day; Q4D, every 4days. FIG. 31C shows that in Colo205 xenografts, increased ERK1/2phosphorylation correlates with BVD-523 concentration.

FIG. 32A shows signaling effects of ERK1/2 inhibitors. Using RPPA,effects on proteins are measured in cell lines (A375, AN3Ca, Colo205,HCT116, HT29 and MIAPaca2) following treatment with ERK1/2 inhibitorsBVD-523 (BVD), Vx11e (Vx), GDC-0994 (GDC), or SCH722984 (SCH). FIG. 32Bshows that the ERK inhibitors BVD-523, GDC-0994, and Vx11e havedifferential effects on phospho-ERK (ERK 1/2 T202 Y204) compared withSCH722984; phospho-RSK (p90 RSK 380) and Cyclin D1 are inhibited by theERK inhibitors tested. Abbreviations: BRAFi, BRAF inhibitors; MEKi, MEKinhibitors. FIG. 32C shows a western blot assay of cellular and nuclearfractions from a RKO cell line following treatment with BVD-523,trametinib, SCH722984, or dabrafenib. Histone H3 (nuclear localizedprotein) and HSP90 (cytoplasmically localized protein) were included aspositive controls to confirm that the nuclear and cytoplasmic fractionswere properly enriched; nuclear fractions have high H3 and cytoplasmicfractions have higher HSP90.

FIG. 33 shows that the ERK inhibitors BVD-523, Vx11, GDC-0994, andSCH772984 (SCH) demonstrate cell line-dependent changes in phospho-ATKlevels. Abbreviation: DMSO, dimethyl sulfoxide.

FIG. 34A-FIG. 34D show that BVD-523 demonstrates activity in models ofresistance to BRAF/MEK inhibition. The appearance of resistance toBVD-523, dabrafenib, or trametinib in BRAF^(V600E) A375 cells followingexposure to increasing concentrations of drug is indicated. A strict setof “criteria” was applied to determine when the dose could be increasedin order to ensure that the kinetics of the acquisition of resistancebetween treatments was comparable. See, Example 1. Time is shown againstmultipliers of IC₅₀; each point on the plotted line represents a changeof medium or cell split. FIG. 34A shows that adapting cells to growth inthe presence of BVD-523 was more challenging than with either dabrafenibor trametinib. FIG. 34B shows that BVD-523 sensitivity is retained inA375 cells cultured to acquire resistance to combined BRAF(dabrafenib)+MEK (trametinib) inhibition. In FIG. 34C, cells weretreated with compound for 96 h and viability was assessed usingCellTiter-Glo®. BVD-523 activity is retained in BRAF^(V600E) RKO cellscross-resistant to BRAF (dabrafenib) and MEK (trametinib) inhibitors dueto endogenous heterozygous knock-in of MEK1^(Q56P). FIG. 34D shows thatBVD-523 inhibition of pRSK in BRAF^(V600E)-mutant cell line RKO ismaintained in the presence of MEK1^(Q56P), which confers resistance toMEK and BRAF inhibition. Knock-in of KRAS mutant alleles into SW48 celllines significantly diminishes sensitivity to the MEK inhibitorstrametinib and selumetinib, while comparatively sensitivity to BVD-523is retained.

FIG. 35A shows BVD-523 in vivo activity in xenografts derived from avemurafenib-relapsed patient. Mean tumor volume (±SEM) is shown forBVD-523 100 mg/kg BID alone, dabrafenib 50 mg/kg BID alone, and BVD-523100 mg/kg BID plus dabrafenib 50 mg/kg BID. Abbreviations: BID, twicedaily; SEM, standard error of mean.

FIG. 36A-FIG. 36D show the benefit of combined BVD-523 and BRAFinhibition. FIG. 36A-FIG. 36B show that the combination of BVD-523 plusdabrafenib exhibited superior antitumor activity compared with treatmentwith either agent alone in a A375 BRAF^(V600E)-melanoma cell linexenograft model with a tumor start volume of 75-144 mm³. FIG. 36C-FIG.36D show similar data from the same model with an enlarged tumor volume(700-800 mm³) at the start of dosing. Plots of mean tumor growth (leftpanels) and Kaplan-Meier survival (right panels) are presented for eachstudy. Abbreviations: BID, twice daily; QD, once daily.

FIG. 37A shows that, in SW48 colorectal cells engineered with KRASalleles, response to paclitaxel was unaltered compared to control. FIG.37B shows combination interactions between BVD-523 and vemurafenib,which were assessed using an 8×10 matrix of concentrations using theLoewe Additivity and Bliss Independence Models, and analyzed withHorizon's Chalice, Bioinformatics Software. Chalice enables potentialsynergistic interactions to be identified by displaying the calculatedexcess inhibition over that predicted as being additive across the dosematrix as a heat map, and by reporting a quantitative “Synergy Score”based on the Loewe model. The results suggest that interactions betweenBVD-523 and vemurafenib are at least additive, and in some casessynergistic in melanoma cell lines carrying a BRAF^(V600E) mutation.FIG. 37C shows that BVD-523 in combination with dabrafenib markedlydelays the onset of acquired resistance in A375 BRAF^(V600E) melanomacells. The temporal acquisition of resistance in response to escalatingconcentrations of dabrafenib alone or in combination with BVD-523 ortrametinib was assessed. Strict criteria were applied as to when thedose could be increased to ensure that the kinetics of adaptation wascomparable between treatments. See, Example 1.

FIG. 38 shows that BVD-523 inhibits ex vivo PMA-stimulated RSK1/2phosphorylation in human whole blood. Averages of BVD-523 concentrationdata set are indicated by (-). n=20 for each concentration of BVD-523.Abbreviations: PBMC, peripheral blood mononuclear cells; RSK, ribosomalS6 kinase.

FIG. 39A shows steady-state BVD-523 pharmacokinetics (Cycle 1, Day 15).The dashed red line indicates an EC₅₀ 200 ng/mL HWB. Abbreviations: AUC,area under the curve; BID, twice daily; C_(max), maximum concentration;EC₅₀, 50% maximum effective concentration; HWB, human whole blood; SD,standard deviation. FIG. 39B shows pharmacodynamic inhibition of ERKphosphorylation by BVD-523 in human whole blood. Abbreviations: BID,twice daily; pRSK, phospho-RSK; RSK, ribosomal S6 kinase.

FIG. 40A shows the best radiographic response in patients treated withBVD-523. Included are all patients with disease measured by RECIST v1.1who received ≥1 dose of study treatment and had >1 on-treatment tumorassessment (25/27; 2 did not receive both scans of target lesions).Response was measured as the change from baseline in the sum of thelongest diameter of each target lesion. Dose shown is that which thepatient was receiving at the time of response. The dashed line indicatesthe threshold for a partial response according to RECIST v1.1.Abbreviations: CRC, colorectal cancer; NET, neuroendocrine tumors;NSCLC, non-small cell lung cancer; NSGCT, nonseminomatous germ celltumors; PNET, pancreatic NET; PTC, papillary thyroid cancer; RECISTv1.1, Response Evaluation Criteria in Solid Tumors version 1.1; SLD, sumof the largest diameter. FIG. 40B shows a computerized tomography scanof a confirmed partial response in a 61-year-old patient with aBRAF-mutant melanoma treated with BVD-523.

FIG. 41 shows tumor response and tumor progression. Shown is a swimmerplot of tumor response, tumor progression, and duration of treatment inresponse-evaluable patients treated with BVD-523. Origin of the verticalaxis corresponds to randomization date or reference start date. Analysiscut-off date: Dec. 1, 2015. Abbreviation: BID, twice daily.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention is a method for treating orameliorating the effects of a cancer in a subject, which cancer isrefractory or resistant to non-ERK MAPK pathway inhibitor therapy. Themethod comprises administering to the subject an effective amount ofBVD-523 or a pharmaceutically acceptable salt thereof.

As used herein, the terms “treat,” “treating,” “treatment” andgrammatical variations thereof mean subjecting an individual subject toa protocol, regimen, process or remedy, in which it is desired to obtaina physiologic response or outcome in that subject, e.g., a patient. Inparticular, the methods and compositions of the present invention may beused to slow the development of disease symptoms or delay the onset ofthe disease or condition, or halt the progression of diseasedevelopment. However, because every treated subject may not respond to aparticular treatment protocol, regimen, process or remedy, treating doesnot require that the desired physiologic response or outcome be achievedin each and every subject or subject population, e.g., patientpopulation. Accordingly, a given subject or subject population, e.g.,patient population may fail to respond or respond inadequately totreatment.

As used herein, the terms “ameliorate”, “ameliorating” and grammaticalvariations thereof mean to decrease the severity of the symptoms of adisease in a subject.

As used herein, a “subject” is a mammal, preferably, a human. Inaddition to humans, categories of mammals within the scope of thepresent invention include, for example, farm animals, domestic animals,laboratory animals, etc. Some examples of farm animals include cows,pigs, horses, goats, etc. Some examples of domestic animals includedogs, cats, etc. Some examples of laboratory animals include primates,rats, mice, rabbits, guinea pigs, etc.

In the present invention, BVD-523 corresponds to a compound according toformula (I):

and pharmaceutically acceptable salts thereof. BVD-523 may besynthesized according to the methods disclosed, e.g., in U.S. Pat. No.7,354,939. Enantiomers and racemic mixtures of both enantiomers ofBVD-523 are also contemplated within the scope of the present invention.BVD-523 is an ERK1/2 inhibitor with a mechanism of action that isbelieved to be, e.g., unique and distinct from certain other ERK1/2inhibitors, such as SCH772984 and the pyrimidinal structure used byHatzivassiliou et al. (2012). For example, other ERK1/2 inhibitors, suchas SCH772984, inhibit autophosphorylation of ERK (Morris et al., 2013),whereas BVD-523 allows for the autophosphorylation of ERK while stillinhibiting ERK. (See, e.g., FIG. 18).

As used herein, the words “resistant” and “refractory” are usedinterchangeably. Being “resistant” to non-ERK MAPK pathway inhibitortherapy treatments means that non-ERK MAPK inhibitors have reducedefficacy in treating cancer.

As used herein, a “non-ERK MAPK inhibitor” means any substance thatreduces the activity, expression or phosphorylation of proteins or othermembers of the MAPK pathway that results in a reduction of cell growthor an increase in cell death, with the exception of ERK1/2 inhibitors.As used herein, an “ERK1/2 inhibitor” means those substances that (i)directly interact with ERK1 and/or ERK2, e.g., by binding to ERK1/2 and(ii) decrease the expression or the activity of ERK1 and/or ERK2 proteinkinases. Therefore, inhibitors that act upstream of ERK1/2, such as MEKinhibitors and RAF inhibitors, are not ERK1/2 inhibitors according tothe present invention (but they are non-ERK MAPK inhibitors).Non-limiting examples of ERK1/2 inhibitors according to the presentinvention include AEZS-131 (Aeterna Zentaris), AEZS-136 (AeternaZentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck& Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.),pharmaceutically acceptable salts thereof, and combinations thereof.

An overview of the mammalian MAPK cascades is shown in FIG. 21. The MAPKpathway is reviewed in e.g., Akinleye et al., 2013. Briefly, withrespect to the ERK1/2 module in FIG. 21 (light purple box), the MAPK 1/2signaling cascade is activated by ligand binding to receptor tyrosinekinases (RTK). The activated receptors recruit and phosphorylate adaptorproteins Grb2 and SOS, which then interact with membrane-bound GTPaseRas and cause its activation. In its activated GTP-bound form, Rasrecruits and activates RAF kinases (A-RAF, B-RAF, and C-RAF/RAF-1). Theactivated RAF kinases activate MAPK 1/2 (MKK1/2), which in turncatalyzes the phosphorylation of threonine and tyrosine residues in theactivation sequence Thr-Glu-Tyr of ERK1/2. With respect to the JNK/p38module (yellow box in FIG. 21), upstream kinases, MAP3Ks, such asMEKK1/4, ASK1/2, and MLK1/2/3, activate MAP2K3/6 (MKK3/6), MAP2K4(MKK4), and MAP2K7 (MKK7). These MAP2K's then activate JNK proteinkinases, including JNK1, JNK2, and JNK3, as well as p38 α/β/γ/δ. Toexecute their functions, JNKs activate several transcription factors,including c-Jun, ATF-2, NF-ATc1, HSF-1 and STAT3. With respect to theERK5 module (blue box in FIG. 21), the kinases upstream of MAP2K5 (MKK5)are MEKK2 and MEKK3. The best characterized downstream target of MEK5 isERK5, also known as big MAP kinase 1 (BMK1) because it is twice the sizeof other MAPKs.

Non-limiting examples of non-ERK MAPK pathway inhibitors according tothe present invention include RAS inhibitors, RAF inhibitors (such as,e.g., inhibitors of A-RAF, B-RAF, C-RAF (RAF-1)), MEK inhibitors, andcombinations thereof. Preferably, the non-ERK MAPK pathway inhibitorsare BRAF inhibitors, MEK inhibitors, and combinations thereof.

As used herein, a “RAS inhibitor” means those substances that (i)directly interact with RAS, e.g., by binding to RAS and (ii) decreasethe expression or the activity of RAS. Non-limiting exemplary RASinhibitors include, but are not limited to, farnesyl transferaseinhibitors (such as, e.g., tipifarnib and lonafarnib), farnesylgroup-containing small molecules (such as, e.g., salirasib andTLN-4601), DCAI, as disclosed by Maurer (Maurer et al., 2012), Kobe0065and Kobe2602, as disclosed by Shima (Shima et al., 2013), HBS 3 (Patgiriet al., 2011), and AIK-4 (Allinky).

As used herein, a “RAF inhibitor” means those substances that (i)directly interact with RAF, e.g., by binding to RAF and (ii) decreasethe expression or the activity of RAF, such as, e.g., A-RAF, B-RAF, andC-RAF (RAF-1). Non-limiting exemplary RAF inhibitors, including BRAFinhibitors, include:

AAL881 (Novartis); AB-024 (Ambit Biosciences), ARQ-736 (ArQule), ARQ-761(ArQule), AZ628 (Axon Medchem BV), BeiGene-283 (BeiGene), BUB-024 (MLN2480) (Sunesis & Takeda), b-raf inhibitor (Sareum), BRAF kinaseinhibitor (Selexagen Therapeutics), BRAF siRNA 313(tacaccagcaagctagatgca) and 523 (cctatcgttagagtcttcctg) (Liu et al.,2007), CTT239065 (Institute of Cancer Research), dabrafenib(GSK2118436), DP-4978 (Deciphera Pharmaceuticals), HM-95573 (Hanmi),GDC-0879 (Genentech), GW-5074 (Sigma Aldrich), ISIS 5132 (Novartis),L779450 (Merck), LBT613 (Novartis), LErafAON (NeoPharm, Inc.), LGX-818(Novartis), pazopanib (GlaxoSmithKline), PLX3202 (Plexxikon), PLX4720(Plexxikon), PLX5568 (Plexxikon), RAF-265 (Novartis), RAF-365(Novartis), regorafenib (Bayer Healthcare Pharmaceuticals, Inc.), RO5126766 (Hoffmann-La Roche), SB-590885 (GlaxoSmithKline), SB699393(GlaxoSmithKline), sorafenib (Onyx Pharmaceuticals), TAK 632 (Takeda),TL-241 (Teligene), vemurafenib (RG7204 or PLX4032) (Daiichi Sankyo),XL-281 (Exelixis), ZM-336372 (AstraZeneca), pharmaceutically acceptablesalts thereof, and combinations thereof.

As used herein, a “MEK inhibitor” means those substances that (i)directly interact with MEK, e.g., by binding to MEK and (ii) decreasethe expression or the activity of MEK. Thus, inhibitors that actupstream of MEK, such as RAS inhibitors and RAF inhibitors, are not MEFinhibitors according to the present invention. Non-limiting examples ofMEK inhibitors include anthrax toxin, antroquinonol (GoldenBiotechnology), ARRY-142886(6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide) (Array BioPharma), ARRY-438162 (ArrayBioPharma), AS-1940477 (Astellas), AS-703988 (Merck KGaA), bentamapimod(Merck KGaA), BI-847325 (Boehringer Ingelheim), E-6201 (Eisai), GDC-0623(Hoffmann-La Roche), GDC-0973 (cobimetinib) (Hoffmann-La Roche), L783277(Merck), lethal factor portion of anthrax toxin, MEK162 (ArrayBioPharma), PD 098059(2-(2′-amino-3′-methoxyphenyl)-oxanaphthalen-4-one) (Pfizer), PD 184352(CI-1040) (Pfizer), PD-0325901 (Pfizer), pimasertib (SantheraPharmaceuticals), RDEA119 (Ardea Biosciences/Bayer), refametinib(AstraZeneca), RG422 (Chugai Pharmaceutical Co.), RO092210 (Roche),RO4987655 (Hoffmann-La Roche), RO5126766 (Hoffmann-La Roche),selumetinib (AZD6244) (AstraZeneca), SL327 (Sigma), TAK-733 (Takeda),trametinib (Japan Tobacco), U0126(1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene) (Sigma),WX-554 (Wilex), YopJ polypeptide (Mittal et al., 2010), pharmaceuticallyacceptable salts thereof, and combinations thereof.

In one aspect of this embodiment, substantially all phosphorylation ofribosomal s6 kinase (RSK) is inhibited after administration of BVD-523or a pharmaceutically acceptable salt thereof. As used herein in thecontext of RSK phosphorylation, “substantially all” means a reduction ofgreater than 50% reduction, preferably greater than 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% Areduction.

In another aspect of this embodiment, the cancer has MAPK activity. Asused herein, having “MAPK activity” means that proteins downstream ofERK are still active, even if proteins upstream of ERK may not beactive. Such a cancer may be a solid tumor cancer or a hematologiccancer.

In the present invention, cancers include both solid and hemotologiccancers. Non-limiting examples of solid cancers include adrenocorticalcarcinoma, anal cancer, bladder cancer, bone cancer (such asosteosarcoma), brain cancer, breast cancer, carcinoid cancer, carcinoma,cervical cancer, colon cancer, endometrial cancer, esophageal cancer,extrahepatic bile duct cancer, Ewing family of cancers, extracranialgerm cell cancer, eye cancer, gallbladder cancer, gastric cancer, germcell tumor, gestational trophoblastic tumor, head and neck cancer,hypopharyngeal cancer, islet cell carcinoma, kidney cancer, largeintestine cancer, laryngeal cancer, leukemia, lip and oral cavitycancer, liver tumor/cancer, lung tumor/cancer, lymphoma, malignantmesothelioma, Merkel cell carcinoma, mycosis fungoides, myelodysplasticsyndrome, myeloproliferative disorders, nasopharyngeal cancer,neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarianepithelial cancer, ovarian germ cell cancer, pancreatic cancer,paranasal sinus and nasal cavity cancer, parathyroid cancer, penilecancer, pituitary cancer, plasma cell neoplasm, prostate cancer,rhabdomyosarcoma, rectal cancer, renal cell cancer, transitional cellcancer of the renal pelvis and ureter, salivary gland cancer, Sezarysyndrome, skin cancers (such as cutaneous t-cell lymphoma, Kaposi'ssarcoma, mast cell tumor, and melanoma), small intestine cancer, softtissue sarcoma, stomach cancer, testicular cancer, thymoma, thyroidcancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer,and Wilms' tumor.

Examples of hematologic cancers include, but are not limited to,leukemias, such as adult/childhood acute lymphoblastic leukemia,adult/childhood acute myeloid leukemia, chronic lymphocytic leukemia,chronic myelogenous leukemia, and hairy cell leukemia, lymphomas, suchas AIDS-related lymphoma, cutaneous T-cell lymphoma, adult/childhoodHodgkin lymphoma, mycosis fungoides, adult/childhood non-Hodgkinlymphoma, primary central nervous system lymphoma, Sezary syndrome,cutaneous T-cell lymphoma, and Waldenstrom macroglobulinemia, as well asother proliferative disorders such as chronic myeloproliferativedisorders, Langerhans cell histiocytosis, multiple myeloma/plasma cellneoplasm, myelodysplastic syndromes, andmyelodysplastic/myeloproliferative neoplasms.

Preferably, the cancer is selected from the group consisting of a cancerof the large intestine, breast cancer, pancreatic cancer, skin cancer,and endometrial cancers. More preferably, the cancer is melanoma.

In another aspect of this embodiment, the method further comprisesadministering to the subject at least one additional therapeutic agenteffective for treating or ameliorating the effects of the cancer. Theadditional therapeutic agent may be selected from the group consistingof an antibody or fragment thereof, a cytotoxic agent, a toxin, aradionuclide, an immunomodulator, a photoactive therapeutic agent, aradiosensitizing agent, a hormone, an anti-angiogenesis agent, andcombinations thereof.

As used herein, an “antibody” encompasses naturally occurringimmunoglobulins as well as non-naturally occurring immunoglobulins,including, for example, single chain antibodies, chimeric antibodies(e.g., humanized murine antibodies), and heteroconjugate antibodies(e.g., bispecific antibodies). Fragments of antibodies include thosethat bind antigen, (e.g., Fab′, F(ab′)₂, Fab, Fv, and rIgG). See also,e.g., Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co.,Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., NewYork (1998). The term antibody also includes bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies. The term “antibody”further includes both polyclonal and monoclonal antibodies.

Examples of therapeutic antibodies that may be used in the presentinvention include rituximab (Rituxan), Cetuximab (Erbitux), bevacizumab(Avastin), and Ibritumomab (Zevalin).

Cytotoxic agents according to the present invention include DNA damagingagents, antimetabolites, anti-microtubule agents, antibiotic agents,etc. DNA damaging agents include alkylating agents, platinum-basedagents, intercalating agents, and inhibitors of DNA replication.Non-limiting examples of DNA alkylating agents include cyclophosphamide,mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide,carmustine, lomustine, streptozocin, busulfan, temozolomide,pharmaceutically acceptable salts thereof, prodrugs, and combinationsthereof. Non-limiting examples of platinum-based agents includecisplatin, carboplatin, oxaliplatin, nedaplatin, satraplatin, triplatintetranitrate, pharmaceutically acceptable salts thereof, prodrugs, andcombinations thereof. Non-limiting examples of intercalating agentsinclude doxorubicin, daunorubicin, idarubicin, mitoxantrone,pharmaceutically acceptable salts thereof, prodrugs, and combinationsthereof. Non-limiting examples of inhibitors of DNA replication includeirinotecan, topotecan, amsacrine, etoposide, etoposide phosphate,teniposide, pharmaceutically acceptable salts thereof, prodrugs, andcombinations thereof. Antimetabolites include folate antagonists such asmethotrexate and premetrexed, purine antagonists such as6-mercaptopurine, dacarbazine, and fludarabine, and pyrimidineantagonists such as 5-fluorouracil, arabinosylcytosine, capecitabine,gemcitabine, decitabine, pharmaceutically acceptable salts thereof,prodrugs, and combinations thereof. Anti-microtubule agents includewithout limitation vinca alkaloids, paclitaxel (Taxol®), docetaxel(Taxotere®), and ixabepilone (Ixempra®). Antibiotic agents includewithout limitation actinomycin, anthracyclines, valrubicin, epirubicin,bleomycin, plicamycin, mitomycin, pharmaceutically acceptable saltsthereof, prodrugs, and combinations thereof.

Cytotoxic agents according to the present invention also include aninhibitor of the PI3K/Akt pathway. Non-limiting examples of an inhibitorof the PI3K/Akt pathway include A-674563 (CAS #552325-73-2), AGL 2263,AMG-319 (Amgen, Thousand Oaks, Calif.), AS-041164(5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850(5-(2,2-Difluoro-benzo[1,3]dioxol-5-ylmethylene)-thiazolidine-2,4-dione),AS-605240 (5-quinoxilin-6-methylene-1,3-thiazolidine-2,4-dione), AT7867(CAS #857531-00-1), benzimidazole series, Genentech (Roche HoldingsInc., South San Francisco, Calif.), BML-257 (CAS #32387-96-5), CAL-120(Gilead Sciences, Foster City, Calif.), CAL-129 (Gilead Sciences),CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (GileadSciences), CAS #612847-09-3, CAS #681281-88-9, CAS #75747-14-7, CAS#925681-41-0, CAS #98510-80-6, CCT128930 (CAS #885499-61-6), CH5132799(CAS #1007207-67-1), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK),FPA 124 (CAS #902779-59-3), GS-1101 (CAL-101) (Gilead Sciences), GSK690693 (CAS #937174-76-0), H-89 (CAS #127243-85-0), Honokiol, IC87114(Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (KarusTherapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1(Karus Therapeutics), KT 5720 (CAS #108068-98-0), Miltefosine, MK-2206dihydrochloride (CAS #1032350-13-2), ML-9 (CAS #105637-50-1),Naltrindole Hydrochloride, OXY-111A (NormOxys Inc., Brighton, Mass.),perifosine, PHT-427 (CAS #1191951-57-1), PI3 kinase delta inhibitor,Merck KGaA (Merck & Co., Whitehouse Station, N.J.), PI3 kinase deltainhibitors, Genentech (Roche Holdings Inc.), PI3 kinase deltainhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hydrabad, India),PI3 kinase delta inhibitors-2, Incozen (Incozen Therapeutics), PI3kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors,Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (RocheHoldings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics(Pathway Therapeutics Ltd., South San Francisco, Calif.), PI3-deltainhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-deltainhibitors, Intellikine (Intellikine Inc., La Jolla, Calif.), PI3-deltainhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.),PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway TherapeuticsLtd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG),PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gammainhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors,Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, PathwayTherapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors,Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitorEvotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gammainhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), pictilisib(Roche Holdings Inc.), PIK-90 (CAS #677338-12-4), SC-103980 (Pfizer, NewYork, N.Y.), SF-1126 (Semafore Pharmaceuticals, Indianapolis, Ind.),SH-5, SH-6, Tetrahydro Curcumin, TG100-115 (Targegen Inc., San Diego,Calif.), Triciribine, X-339 (Xcovery, West Palm Beach, Fla.), XL-499(Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof,and combinations thereof.

In the present invention, the term “toxin” means an antigenic poison orvenom of plant or animal origin. An example is diphtheria toxin orportions thereof.

In the present invention, the term “radionuclide” means a radioactivesubstance administered to the patient, e.g., intravenously or orally,after which it penetrates via the patient's normal metabolism into thetarget organ or tissue, where it delivers local radiation for a shorttime. Examples of radionuclides include, but are not limited to, 1-125,At-211, Lu-177, Cu-67, I-131, Sm-153, Re-186, P-32, Re-188, In-114m, andY-90.

In the present invention, the term “immunomodulator” means a substancethat alters the immune response by augmenting or reducing the ability ofthe immune system to produce antibodies or sensitized cells thatrecognize and react with the antigen that initiated their production.Immunomodulators may be recombinant, synthetic, or natural preparationsand include cytokines, corticosteroids, cytotoxic agents, thymosin, andimmunoglobulins. Some immunomodulators are naturally present in thebody, and certain of these are available in pharmacologic preparations.Examples of immunomodulators include, but are not limited to,granulocyte colony-stimulating factor (G-CSF), interferons, imiquimodand cellular membrane fractions from bacteria, IL-2, IL-7, IL-12, CCL3,CCL26, CXCL7, and synthetic cytosine phosphate-guanosine (CpG).

In the present invention, the term “photoactive therapeutic agent” meanscompounds and compositions that become active upon exposure to light.Certain examples of photoactive therapeutic agents are disclosed, e.g.,in U.S. Patent Application Serial No. 2011/0152230 A1, “PhotoactiveMetal Nitrosyls For Blood Pressure Regulation And Cancer Therapy.”

In the present invention, the term “radiosensitizing agent” means acompound that makes tumor cells more sensitive to radiation therapy.Examples of radiosensitizing agents include misonidazole, metronidazole,tirapazamine, and trans sodium crocetinate.

In the present invention, the term “hormone” means a substance releasedby cells in one part of a body that affects cells in another part of thebody. Examples of hormones include, but are not limited to,prostaglandins, leukotrienes, prostacyclin, thromboxane, amylin,antimullerian hormone, adiponectin, adrenocorticotropic hormone,angiotensinogen, angiotensin, vasopressin, atriopeptin, brainnatriuretic peptide, calcitonin, cholecystokinin,corticotropin-releasing hormone, encephalin, endothelin, erythropoietin,follicle-stimulating hormone, galanin, gastrin, ghrelin, glucagon,gonadotropin-releasing hormone, growth hormone-releasing hormone, humanchorionic gonadotropin, human placental lactogen, growth hormone,inhibin, insulin, somatomedin, leptin, liptropin, luteinizing hormone,melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreaticpolypeptide, parathyroid hormone, prolactin, prolactin releasinghormone, relaxin, renin, secretin, somatostain, thrombopoietin,thyroid-stimulating hormone, testosterone, dehydroepiandrosterone,androstenedione, dihydrotestosterone, aldosterone, estradiol, estrone,estriol, cortisol, progesterone, calcitriol, and calcidiol.

Some compounds interfere with the activity of certain hormones or stopthe production of certain hormones. These hormone-interfering compoundsinclude, but are not limited to, tamoxifen (Nolvadex®), anastrozole(Arimidex®), letrozole (Femara®), and fulvestrant (Faslodex®). Suchcompounds are also within the meaning of hormone in the presentinvention.

As used herein, an “anti-angiogenesis” agent means a substance thatreduces or inhibits the growth of new blood vessels, such as, e.g., aninhibitor of vascular endothelial growth factor (VEGF) and an inhibitorof endothelial cell migration. Anti-angiogenesis agents include withoutlimitation 2-methoxyestradiol, angiostatin, bevacizumab,cartilage-derived angiogenesis inhibitory factor, endostatin, IFN-α,IL-12, itraconazole, linomide, platelet factor-4, prolactin, SU5416,suramin, tasquinimod, tecogalan, tetrathiomolybdate, thalidomide,thrombospondin, thrombospondin, TNP-470, ziv-aflibercept,pharmaceutically acceptable salts thereof, prodrugs, and combinationsthereof.

Another embodiment of the present invention is a method for treating orameliorating the effects of a cancer in a subject. The method comprises:

(a) identifying a subject with cancer that has become refractory orresistant to BRAF inhibitor therapy, MEK inhibitor therapy, or BRAF andMEK inhibitor therapy; and

(b) administering to the subject with said refractory or resistantcancer an effective amount of an ERK inhibitor, which is BVD-523 or apharmaceutically acceptable salt thereof.

Suitable and preferred subjects are as disclosed herein. In thisembodiment, the methods may be used to treat the cancers disclosedabove. In accordance with the present invention, the cancer may haveMAPK activity.

In one aspect of this embodiment, identifying a subject with cancer thatis refractory or resistant to BRAF and/or MEK inhibitor therapycomprises:

(a) obtaining a biological sample from the subject; and

(b) screening the sample to determine whether the subject has becomeresistant to an inhibitor therapy selected from the group consisting ofBRAF inhibitor therapy, MEK inhibitor therapy, and combinations thereof.

In the present invention, biological samples include, but are notlimited to, blood, plasma, urine, skin, saliva, and biopsies. Biologicalsamples are obtained from a subject by routine procedures and methodswhich are known in the art.

Preferably, screening for a cancer that is refractory or resistant toBRAF inhibitor therapy may comprise, e.g., identifying (i) a switchbetween RAF isoforms, (ii) upregulation of RTK or NRAS signaling, (iii)reactivation of mitogen activated protein kinase (MAPK) signaling, (iv)the presence of a MEK activating mutation, and combinations thereof.

A switch between RAF isoforms may occur in subjects having acquiredresistance to BRAF inhibitor therapy. To detect such a switch, BRAFinhibitor-resistant tumor cells may be retrieved from a patient andanalyzed via Western blotting for ERK and phospho-ERK levels in thepresence of a BRAF inhibitor. Comparison with BRAF inhibitor-sensitivecells treated with a BRAF inhibitor may reveal higher levels ofphospho-ERK in BRAF inhibitor-resistant tumor cells, implying that aswitch has taken place in which another RAF isoform phosphorylates ERKin place of BRAF. Confirmation of which RAF isoform has taken over mayinvolve sh/siRNA-mediated knockdown of ARAF and CRAF individually inBRAF inhibitor-resistant cells exposed to a BRAF inhibitor, followed bysubsequent Western blotting for ERK and phospho-ERK levels. If, forexample, ARAF knockdown in BRAF inhibitor-resistant cells exposed to aBRAF inhibitor still results in high levels of phospho-ERK, it wouldindicate that CRAF has taken over phosphorylating ERK. Likewise, if CRAFwas knocked down in BRAF inhibitor-resistant cells exposed to BRAFinhibitor and ERK was still highly phosphorylated, it would mean thatARAF has taken over ERK phosphorylation. RAF isoform switching may alsoinvolve simultaneous knockdown of ARAF and CRAF in BRAFinhibitor-resistant cells in the presence of BRAF inhibitor, effectivelyblocking all RAF-mediated phosphorylation. A resulting decrease in ERKphosphorylation would indicate that the BRAF inhibitor-resistant cellshave the capacity to switch between RAF isoforms in order tophosphorylate ERK (Villanueva, et al., 2010).

Upregulation of RTK or NRAS signaling may also be a cause of BRAFinhibitor resistance. Detection may, e.g., first involve using Westernblotting protocols with phospho-specific antibodies to analyze theactivation of the downstream RAF effectors MEK1/2 and ERK1/2. If BRAFinhibitor-resistant cells show high activation levels of these proteinsin the presence of a BRAF inhibitor, RTK or NRAS upregulation may be thecause. Gene expression profiling (or other related methods) of BRAFinhibitor-resistant cells in the presence of a BRAF inhibitor may revealhigher expression levels of KIT, MET, EGFR, and PDGFRβ RTKs as comparedto BRAF inhibitor-sensitive cells. Real-time quantitative polymerasechain reaction experiments, or other similar procedures, focusing on anyof these genes may confirm higher expression levels while phospho-RTKarrays (R&D Systems, Minneapolis, Minn.) may show elevatedactivation-associated tyrosine phosphorylation. Alternatively, NRASactivation may be detected by various gene sequencing protocols.Activating mutations in NRAS, particularly Q61K, may indicate that B-RAFsignaling has been bypassed. In melanoma cells, activated NRAS usesC-RAF to signal to MEK-ERK. Thus, activated NRAS may enable a similarbypass pathway in BRAF inhibitor-resistant cells exposed to BRAFinhibitor. Further confirmation of these mechanisms in a given BRAFinhibitor-resistant sample may be accomplished, for example, usingsh/siRNA-mediated knockdown of upregulated RTKs or activated NRAS in thepresence of BRAF inhibitor. Any significant levels of growth inhibitionmay indicate that upregulation of RTK or NRAS signaling is the cause ofBRAF inhibition in that particular sample (Nazarian, et al., 2010).

Detecting reactivation of MAPK signaling in BRAF inhibitor-resistantcells may indicate another bypass mechanism for BRAF inhibitorresistance. COT and C-RAF have been shown to be upregulated in a BRAFV600E background exposed to BRAF inhibitor. Quantiative real-timeRT-PCR, e.g., may reveal increased COT expression in BRAFinhibitor-resistant cells in the presence of BRAF inhibitor.Furthermore, sh/siRNA-mediated knockdown of COT in BRAFinhibitor-resistant cells in the presence of BRAF inhibitor may reducethe viability of BRAF inhibitor-resistant cells, indicating that theseparticular cells may be sensitive to COT inhibition and/or combinationBRAF inhibitor/MEK inhibitor treatments (Johannessen, et al., 2010).

Reactivation of MAPK signaling may also be accomplished in a BRAFinhibitor-resistant background by activating mutations in MEK1.Targeted, massively parallel sequencing of genomic DNA from a BRAFinhibitor-resistant tumor may reveal activating mutations in MEK1, suchas C121S, G128D, N122D, and Y130, among others. Other, undocumentedmutations in MEK1 may be analyzed by, for example, expressing theparticular mutation in a BRAF inhibitor-sensitive cell line such asA375. Determining levels of growth inhibition in these cells uponexposure to BRAF inhibitor may indicate if the MEK1 mutation is causingresistance to BRAF inhibitory therapy. To confirm such a finding,Western blotting for elevated levels of phospho-ERK1/2 in cellsectopically expressing the MEK1 mutation may indicate that the MEK1mutation is allowing the BRAF inhibitor-resistant tumor to bypass BRAFand promote phosphorylation of ERK through MEK1 (Wagle, et al., 2011).

In accordance with the present invention, screening for a cancer that isrefractory or resistant to MEK inhibitor therapy may comprise, e.g.,identifying (i) amplification of mutant BRAF, (ii) STAT3 upregulation,(iii) mutations in the allosteric pocket of MEK that directly blockbinding of inhibitors to MEK or lead to constitutive MEK activity, andcombinations thereof.

Amplification of mutant BRAF may cause MEK inhibitor resistance. MEKinhibitor resistance is typically associated with high levels ofphosphorylated ERK and MEK in the presence of a MEK inhibitor, which maybe assessed via, for example, Western blotting. Amplification of mutantBRAF in MEK inhibitor-resistant cell lines may be detected by, forexample, fluorescence in situ hybridization (FISH) or quantitative PCRfrom genomic DNA of the resistant cell lines. Confirmation that BRAFamplification is a primary cause of MEK inhibitor resistance may entailusing BRAF-targeted sh/siRNAs in resistant cells. If a significantdecrease in MEK or ERK phosphorylation is observed, BRAF amplificationmay be a suitable target for further therapeutic approaches. (Corcoran,et al., 2010).

Identifying STAT3 upregulation may indicate that a particular tumorsample is resistant to MEK inhibitor therapy. Genome-wide expressionprofiling may reveal the STAT3 pathway to be upregulated in a tumor.Other techniques, such as Western blotting for phospho-STAT3 andreal-time qPCR for the STAT pathway-associated genes JAK1 and IL6ST mayreveal upregulated STAT3. Further confirmation that STAT3 upregulationcauses MEK inhibitor resistance in a particular sample may comprise theuse of sh/siRNAs against STAT3 in the sample followed by appropriateWestern blotting for MEK and ERK activation as well as phospho-STAT3 andtotal STAT3. Growth inhibition studies may show that STAT3 knockdownsensitizes previously MEK inhibitor-resistant cells to MEK inhibition. Asimilar effect may be seen if the sample were exposed to a STAT3inhibitor such as JSI-124. Additional confirmation that STAT3upregulation is the cause of MEK inhibitor resistance in a particulartumor could arise from Western blotting for BIM expression, includingBIM-EL, BIM-L, and BIM-SL. BIM expression leads to MEK inhibitor-inducedapoptosis, thus STAT3 upregulation may lower BIM levels. STAT3 is knownto regulate the expression of miR 17-92, which suppresses BIMexpression. Upregulated STAT3 may lead to higher levels of miR 17-92,which will lower BIM levels and promote resistance to MEK inhibition.Thus, real-time qPCR of miR 17-92 levels may also assist in assessingwhether STAT3 upregulation is causing MEK inhibition resistance in aparticular sample. (Dai, et al., 2011).

Mutations in the allosteric pocket of MEK that can directly blockbinding of inhibitors to MEK or lead to constitutive MEK activity may bedetected by methods disclosed below. Such mutations have been identifiedpreviously by Emery and colleagues (Emery, et al., 2009) as well as Wangand colleagues (Wang et al., 2011). Other mutations may affect MEK1codons located within or abutting the N-terminal negative regulatoryhelix, such as P124L and Q56P. (Id.).

Methods for identifying mutations in nucleic acids, such as the aboveidentified MEK genes, are known in the art. Nucleic acids may beobtained from biological samples. In the present invention, biologicalsamples include, but are not limited to, blood, plasma, urine, skin,saliva, and biopsies. Biological samples are obtained from a subject byroutine procedures and methods which are known in the art.

Non-limiting examples of methods for identifying mutations include PCR,sequencing, hybrid capture, in-solution capture, molecular inversionprobes, fluorescent in situ hybridization (FISH) assays, andcombinations thereof.

Various sequencing methods are known in the art. These include, but arenot limited to, Sanger sequencing (also referred to as dideoxysequencing) and various sequencing-by-synthesis (SBS) methods asdisclosed in, e.g., Metzker 2005, sequencing by hybridization, byligation (for example, WO 2005021786), by degradation (for example, U.S.Pat. Nos. 5,622,824 and 6,140,053) and nanopore sequencing (which iscommercially available from Oxford Nanopore Technologies, UK). In deepsequencing techniques, a given nucleotide in the sequence is read morethan once during the sequencing process. Deep sequencing techniques aredisclosed in e.g., U.S. Patent Publication No. 20120264632 andInternational Patent Publication No. WO2012125848.

PCR-based methods for detecting mutations are known in the art andemploy PCR amplification, where each target sequence in the sample has acorresponding pair of unique, sequence-specific primers. For example,the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) method allows for rapid detection of mutations after thegenomic sequences are amplified by PCR. The mutation is discriminated bydigestion with specific restriction endonucleases and is identified byelectrophoresis. See, e.g., Ota et al., 2007. Mutations may also bedetected using real time PCR. See, e.g., International Applicationpublication No. WO2012046981.

Hybrid capture methods are known in the art and are disclosed in e.g.,U.S. Patent Publication No. 20130203632 and U.S. Pat. Nos. 8,389,219 and8,288,520. These methods are based on the selective hybridization of thetarget genomic regions to user-designed oligonucleotides. Thehybridization can be to oligonucleotides immobilized on high or lowdensity microarrays (on-array capture), or solution-phase hybridizationto oligonucleotides modified with a ligand (e.g. biotin) which cansubsequently be immobilized to a solid surface, such as a bead(in-solution capture).

Molecular Inversion Probe (MIP) techniques are known in the art and aredisclosed in e.g., Absalan et al., 2008. This method uses MIP molecules,which are special “padlock” probes (Nilsson et al, 1994) for genotyping.A MIP molecule is a linear oligonucleotide that contains specificregions, universal sequences, restriction sites and a Tag (index)sequence (16-22 bp). A MIP hybridizes directly around the geneticmarker/SNP of interest. The MIP method may also use a number of“padlock” probe sets that hybridize to genomic DNA in parallel(Hardenbol et al., 2003). In case of a perfect match, genomic homologyregions are ligated by undergoing an inversion in configuration (assuggested by the name of the technique) and creating a circularmolecule. After the first restriction, all molecules are amplified withuniversal primers. Amplicons are restricted again to ensure shortfragments for hybridization on a microarray. Generated short fragmentsare labeled and, through a Tag sequence, hybridized to a cTag(complementary strand for index) on an array. After the formation ofTag-cTag duplex, a signal is detected.

The following Tables 1, 2, and 3 show the SEQ ID Nos. of representativenucleic acid and amino acid sequences of wild type BRAF, N-RAS, and MEK1from various animals in the sequence listing. These sequences may beused in methods for identifying subjects with mutant BRAF, N-RAS, andMEK1 genotypes.

TABLE 1 BRAF sequences polypeptide or nucleic acid Other SEQ ID NO.sequence Organism information 1 nucleic acid human 2 polypeptide human 3nucleic acid rat (Rattus norvegicus) 4 polypeptide rat (Rattusnorvegicus) 5 nucleic acid mouse, Mus musculus 6 polypeptide mouse, Musmusculus 7 nucleic acid rabbit, Oryctolagus cuniculus 8 polypeptiderabbit, Oryctolagus cuniculus 9 nucleic acid guinea pig, Cavia porcellus10 polypeptide guinea pig, Cavia porcellus 11 nucleic acid dog, Canislupus variant x1 familiaris 12 polypeptide dog, Canis lupus variant x1familiaris 13 nucleic acid dog, Canis lupus variant x2 familiaris 14polypeptide dog, Canis lupus variant x2 familiaris 15 nucleic acid cat,Felis catus 16 polypeptide cat, Felis catus 17 nucleic acid cow, Bostaurus variant X1 18 polypeptide cow, Bos taurus variant X1 19 nucleicacid cow, Bos taurus variant X2 20 polypeptide cow, Bos taurus variantX2 21 nucleic acid cow, Bos taurus variant X3 22 polypeptide cow, Bostaurus variant X3 23 nucleic acid cow, Bos taurus variant X4 24polypeptide cow, Bos taurus variant X4 25 nucleic acid cow, Bos taurusvariant X5 26 polypeptide cow, Bos taurus variant X5 27 nucleic acidcow, Bos taurus variant X6 28 polypeptide cow, Bos taurus variant X6 29nucleic acid cow, Bos taurus variant X7 30 polypeptide cow, Bos taurusvariant X7 31 nucleic acid cow, Bos taurus variant X8 32 polypeptidecow, Bos taurus variant X8 33 nucleic acid cow, Bos taurus variant X9 34polypeptide cow, Bos taurus variant X9 35 nucleic acid cow, Bos taurusvariant X10 36 polypeptide cow, Bos taurus variant X10 37 nucleic acidcow, Bos taurus variant X11 38 polypeptide cow, Bos taurus variant X1139 nucleic acid cow, Bos taurus variant 2 40 polypeptide cow, Bos taurusvariant 2 41 nucleic acid horse, Equus caballus 42 polypeptide horse,Equus caballus 43 nucleic acid chicken, Gallus gallus 44 polypeptidechicken, Gallus gallus

TABLE 2 N-RAS sequences polypeptide or nucleic acid Other SEQ ID NO.sequence Organism information 45 nucleic acid human 46 polypeptide human47 nucleic acid rat (Rattus norvegicus) 48 polypeptide rat (Rattusnorvegicus) 49 nucleic acid mouse, Mus musculus 50 polypeptide mouse,Mus musculus 51 nucleic acid guinea pig, Cavia porcellus 52 polypeptideguinea pig, Cavia porcellus 53 nucleic acid guinea pig, Cavia porcellusvariant X1 54 polypeptide guinea pig, Cavia porcellus variant X1 55nucleic acid dog, Canis lupus familiaris 56 polypeptide dog, Canis lupusfamiliaris 57 nucleic acid cat, Felis catus 58 polypeptide cat, Feliscatus 59 nucleic acid cow, Bos taurus 60 polypeptide cow, Bos taurus 61nucleic acid chicken, Gallus gallus 62 polypeptide chicken, Gallusgallus

TABLE 3 MEK1 sequences polypeptide or nucleic acid SEQ ID NO. sequenceOrganism 63 nucleic acid human 64 polypeptide human 65 nucleic acid rat(Rattus norvegicus) 66 polypeptide rat (Rattus norvegicus) 67 nucleicacid mouse, Mus musculus 68 polypeptide mouse, Mus musculus 69 nucleicacid rabbit, Oryctolagus cuniculus 70 polypeptide rabbit, Oryctolaguscuniculus 71 nucleic acid guinea pig, Cavia porcellus 72 polypeptideguinea pig, Cavia porcellus 73 nucleic acid dog, Canis lupus familiaris74 polypeptide dog, Canis lupus familiaris 75 nucleic acid cat, Feliscatus 76 polypeptide cat, Felis catus 77 nucleic acid cow, Bos taurus 78polypeptide cow, Bos taurus 79 nucleic acid horse, Equus caballus 80polypeptide horse, Equus caballus 81 nucleic acid chicken, Gallus gallus82 polypeptide chicken, Gallus gallus

In another aspect of this embodiment, the method further comprisesadministering at least one additional therapeutic agent, preferably aninhibitor of the PI3K/Akt pathway, as disclosed herein.

A further embodiment of the present invention is a method for treatingor ameliorating the effects of cancer in a subject, which cancer isrefractory or resistant to BRAF inhibitor therapy, MEK inhibitortherapy, or both. The method comprises administering to the subject aneffective amount of BVD-523 or a pharmaceutically acceptable saltthereof.

Suitable and preferred subjects are as disclosed herein. In thisembodiment, the methods may be used to treat the cancers disclosedabove, including those cancers with the mutational backgrounds,resistance profiles, and MAPK activity identified above. Methods ofidentifying such mutations are also as set forth above.

In a further aspect of this embodiment, the method further comprisesadministering to the subject at least one additional therapeutic agent,preferably an inhibitor of the PI3K/Akt pathway, as disclosed herein.

Another embodiment of the present invention is a method for identifyinga subject having cancer who would benefit from therapy with an ERKinhibitor. The method comprises:

(a) obtaining a biological sample from the subject; and

(b) screening the sample to determine whether the subject has one ormore of the following markers:

-   -   (i) a switch between RAF isoforms,    -   (ii) upregulation of RTK or NRAS signaling,    -   (iii) reactivation of mitogen activated protein kinase (MAPK)        signaling,    -   (iv) the presence of a MEK activating mutation,    -   (v) amplification of mutant BRAF,    -   (vi) STAT3 upregulation,    -   (vii) mutations in the allosteric pocket of MEK that directly        block binding of inhibitors to MEK or lead to constitutive MEK        activity,        wherein the presence of one or more of the markers confirms that        the subject's cancer is refractory or resistant to BRAF and/or        MEK inhibitor therapy and that the subject would benefit from        therapy with an ERK inhibitor, which is BVD-523 or a        pharmaceutically acceptable salt thereof.

Suitable and preferred subjects are as disclosed herein. In thisembodiment, the methods may be used to identify a subject having cancersdisclosed above, including those cancers with the mutationalbackgrounds, resistance profiles, and MAPK activity identified above.Methods of identifying such mutations are also as set forth above.

In one aspect of this embodiment, the method further comprisesadministering BVD-523 or a pharmaceutically acceptable salt thereof to asubject having one or more of the markers. Preferably, the methodadditionally comprises administering to the subject having one or moreof the markers at least one additional therapeutic agent, preferably aninhibitor of the PI3K/Akt pathway, as disclosed herein.

An additional embodiment of the present invention is a pharmaceuticalcomposition for treating or ameliorating the effects of a cancer in asubject, which cancer is refractory or resistant to non-ERK MAPK pathwaytherapy. The composition comprises a pharmaceutically acceptable carrieror diluent and an effective amount of BVD-523 or a pharmaceuticallyacceptable salt thereof.

Suitable and preferred subjects and types of non-ERK MAPK pathwayinhibitor therapy are as disclosed herein. In this embodiment, thepharmaceutical composition may be used to treat the cancers disclosedabove, including those cancers with the mutational backgrounds,resistance profiles, and MAPK activity identified above. Methods ofidentifying such mutations are also as set forth above.

In one aspect of this embodiment, the pharmaceutical composition furthercomprises at least one additional therapeutic agent, preferably aninhibitor of the PI3K/Akt pathway, as disclosed herein.

Another embodiment of the present invention is a kit for treating orameliorating the effects of a cancer in a subject, which cancer isrefractory or resistant to non-ERK MAPK pathway therapy. This kitcomprises any pharmaceutical composition according to the presentinvention packaged together with instructions for its use.

The kits may also include suitable storage containers, e.g., ampules,vials, tubes, etc., for each pharmaceutical composition and otherreagents, e.g., buffers, balanced salt solutions, etc., for use inadministering the pharmaceutical compositions to subjects. Thepharmaceutical compositions and other reagents may be present in thekits in any convenient form, such as, e.g., in a solution or in a powderform. The kits may further include a packaging container, optionallyhaving one or more partitions for housing the pharmaceutical compositionand other optional reagents.

Suitable and preferred subjects and types of non-ERK MAPK pathwayinhibitor therapy are as disclosed herein. In this embodiment, the kitmay be used to treat the cancers disclosed above, including thosecancers with the mutational backgrounds, resistance profiles, and MAPKactivity identified herein. Methods of identifying such mutations are asset forth above.

In one aspect of this embodiment, the kit further comprises at least oneadditional therapeutic agent, preferably an inhibitor of the PI3K/Aktpathway, as disclosed herein.

Another embodiment of the present invention is a method for inhibitingphosphorylation of RSK in a cancer cell that is refractory or resistantto a non-ERK MAPK pathway inhibitor. The method comprises contacting thecancer cell with an effective amount of BVD-523 or a pharmaceuticallyacceptable salt thereof for a period of time sufficient forphosphorylation of RSK in the cancer cell to be inhibited. In thisembodiment, “contacting” means bringing BVD-523 or a pharmaceuticallyacceptable salt thereof and optionally one or more additionaltherapeutic agents into close proximity to the cancer cells. This may beaccomplished using conventional techniques of drug delivery to mammals,or in the in vitro situation by, e.g., providing BVD-523 or apharmaceutically acceptable salt thereof and optionally othertherapeutic agents to a culture media in which the cancer cells arelocated. In the ex vivo situation, contacting may be carried out by,e.g., providing BVD-523 or a pharmaceutically acceptable salt thereofand optionally other therapeutic agents to a cancerous tissue.

Suitable and preferred types of non-ERK MAPK pathway inhibitors are asdisclosed herein. In this embodiment, effecting cancer cell death may beaccomplished in cancer cells having various mutational backgrounds,resistance profiles, and MAPK activity as disclosed above. Methods ofidentifying such mutations are also as set forth above.

The methods of this embodiment, which may be carried out in vitro, exvivo, or in vivo, may be used to effect cancer cell death, by e.g.,killing cancer cells, in cells of the types of cancer disclosed herein.

In one aspect of this embodiment, greater than 50% of RSKphosphorylation is inhibited. In another aspect of this embodiment,greater than 75% of RSK phosphorylation is inhibited. In an additionalaspect of this embodiment, greater than 90% of RSK phosphorylation isinhibited. In a further aspect of this embodiment, greater than 95% ofRSK phosphorylation is inhibited. In another aspect of this embodiment,greater than 99% of RSK phosphorylation is inhibited. In an additionalaspect of this embodiment, 100% of RSK phosphorylation is inhibited.

In a further aspect of this embodiment, the cancer cell is a mammaliancancer cell. Preferably, the mammalian cancer cell is obtained from amammal selected from the group consisting of humans, primates, farmanimals, and domestic animals. More preferably, the mammalian cancercell is a human cancer cell.

In a further aspect of this embodiment, the contacting step comprisesadministering BVD-523 or a pharmaceutically acceptable salt to a subjectfrom whom the cancer cell was obtained.

In the present invention, an “effective amount” or a “therapeuticallyeffective amount” of a compound or composition disclosed herein is anamount of such compound or composition that is sufficient to effectbeneficial or desired results as described herein when administered to asubject. Effective dosage forms, modes of administration, and dosageamounts may be determined empirically, and making such determinations iswithin the skill of the art. It is understood by those skilled in theart that the dosage amount will vary with the route of administration,the rate of excretion, the duration of the treatment, the identity ofany other drugs being administered, the age, size, and species ofmammal, e.g., human patient, and like factors well known in the arts ofmedicine and veterinary medicine. In general, a suitable dose of acompound or composition according to the invention will be that amountof the composition, which is the lowest dose effective to produce thedesired effect. The effective dose of a compound or composition of thepresent invention may be administered as two, three, four, five, six ormore sub-doses, administered separately at appropriate intervalsthroughout the day.

A suitable, non-limiting example of a dosage of a BVD-523 and otheranti-cancer agents disclosed herein is from about 1 mg/kg to about 2400mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day,75 mg/kg per day to about 300 mg/kg per day, including from about 1mg/kg to about 100 mg/kg per day. Other representative dosages of suchagents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg,175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kgper day. The effective dose of BVD-523 and other anti-cancer agentsdisclosed herein, may be administered as two, three, four, five, six ormore sub-doses, administered separately at appropriate intervalsthroughout the day.

The BVD-523, other inhibitors, and various other anti-cancer agentsdisclosed herein, or a pharmaceutical composition of the presentinvention may be administered in any desired and effective manner: fororal ingestion, or as an ointment or drop for local administration tothe eyes, or for parenteral or other administration in any appropriatemanner such as intraperitoneal, subcutaneous, topical, intradermal,inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular,intravenous, intraarterial, intrathecal, or intralymphatic. Further,BVD-523, other inhibitors, and various other anti-cancer agentsdisclosed herein, or a pharmaceutical composition of the presentinvention may be administered in conjunction with other treatments.BVD-523, other inhibitors, and various other anti-cancer agentsdisclosed herein, or a pharmaceutical composition of the presentinvention may be encapsulated or otherwise protected against gastric orother secretions, if desired.

The pharmaceutical compositions of the invention comprise one or moreactive ingredients in admixture with one or morepharmaceutically-acceptable diluents or carriers and, optionally, one ormore other compounds, drugs, ingredients and/or materials. Regardless ofthe route of administration selected, the agents/compounds of thepresent invention are formulated into pharmaceutically-acceptable dosageforms by conventional methods known to those of skill in the art. See,e.g., Remington, The Science and Practice of Pharmacy (21^(st) Edition,Lippincott Williams and Wilkins, Philadelphia, Pa.).

Pharmaceutically acceptable diluents or carriers are well known in theart (see, e.g., Remington, The Science and Practice of Pharmacy (21^(st)Edition, Lippincott Williams and Wilkins, Philadelphia, Pa.) and TheNational Formulary (American Pharmaceutical Association, Washington,D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, andsorbitol), starches, cellulose preparations, calcium phosphates (e.g.,dicalcium phosphate, tricalcium phosphate and calcium hydrogenphosphate), sodium citrate, water, aqueous solutions (e.g., saline,sodium chloride injection, Ringer's injection, dextrose injection,dextrose and sodium chloride injection, lactated Ringer's injection),alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol),polyols (e.g., glycerol, propylene glycol, and polyethylene glycol),organic esters (e.g., ethyl oleate and tryglycerides), biodegradablepolymers (e.g., polylactide-polyglycolide, poly(orthoesters), andpoly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils(e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut),cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones,talc, silicylate, etc. Each pharmaceutically acceptable diluent orcarrier used in a pharmaceutical composition of the invention must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not injurious to the subject. Diluents orcarriers suitable for a selected dosage form and intended route ofadministration are well known in the art, and acceptable diluents orcarriers for a chosen dosage form and method of administration can bedetermined using ordinary skill in the art.

The pharmaceutical compositions of the invention may, optionally,contain additional ingredients and/or materials commonly used inpharmaceutical compositions. These ingredients and materials are wellknown in the art and include (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, suchas carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, suchas glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,sodium starch glycolate, cross-linked sodium carboxymethyl cellulose andsodium carbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such astalc, calcium stearate, magnesium stearate, solid polyethylene glycols,and sodium lauryl sulfate; (10) suspending agents, such as ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth; (11) buffering agents; (12) excipients, such as lactose,milk sugars, polyethylene glycols, animal and vegetable fats, oils,waxes, paraffins, cocoa butter, starches, tragacanth, cellulosederivatives, polyethylene glycol, silicones, bentonites, silicic acid,talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, andpolyamide powder; (13) inert diluents, such as water or other solvents;(14) preservatives; (15) surface-active agents; (16) dispersing agents;(17) control-release or absorption-delaying agents, such ashydroxypropylmethyl cellulose, other polymer matrices, biodegradablepolymers, liposomes, microspheres, aluminum monostearate, gelatin, andwaxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)emulsifying and suspending agents; (22), solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan; (23)propellants, such as chlorofluorohydrocarbons and volatile unsubstitutedhydrocarbons, such as butane and propane; (24) antioxidants; (25) agentswhich render the formulation isotonic with the blood of the intendedrecipient, such as sugars and sodium chloride; (26) thickening agents;(27) coating materials, such as lecithin; and (28) sweetening,flavoring, coloring, perfuming and preservative agents. Each suchingredient or material must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the subject. Ingredients and materials suitable for aselected dosage form and intended route of administration are well knownin the art, and acceptable ingredients and materials for a chosen dosageform and method of administration may be determined using ordinary skillin the art.

The pharmaceutical compositions of the present invention suitable fororal administration may be in the form of capsules, cachets, pills,tablets, powders, granules, a solution or a suspension in an aqueous ornon-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, anelixir or syrup, a pastille, a bolus, an electuary or a paste. Theseformulations may be prepared by methods known in the art, e.g., by meansof conventional pan-coating, mixing, granulation or lyophilizationprocesses.

Solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like) may be prepared, e.g., bymixing the active ingredient(s) with one or morepharmaceutically-acceptable diluents or carriers and, optionally, one ormore fillers, extenders, binders, humectants, disintegrating agents,solution retarding agents, absorption accelerators, wetting agents,absorbents, lubricants, and/or coloring agents. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using a suitable excipient. A tablet may be made by compressionor molding, optionally with one or more accessory ingredients.Compressed tablets may be prepared using a suitable binder, lubricant,inert diluent, preservative, disintegrant, surface-active or dispersingagent. Molded tablets may be made by molding in a suitable machine. Thetablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient therein.They may be sterilized by, for example, filtration through abacteria-retaining filter. These compositions may also optionallycontain opacifying agents and may be of a composition such that theyrelease the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.The active ingredient can also be in microencapsulated form.

Liquid dosage forms for oral administration includepharmaceutically-acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. The liquid dosage forms may containsuitable inert diluents commonly used in the art. Besides inertdiluents, the oral compositions may also include adjuvants, such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents. Suspensions maycontain suspending agents.

The pharmaceutical compositions of the present invention for rectal orvaginal administration may be presented as a suppository, which may beprepared by mixing one or more active ingredient(s) with one or moresuitable nonirritating diluents or carriers which are solid at roomtemperature, but liquid at body temperature and, therefore, will melt inthe rectum or vaginal cavity and release the active compound. Thepharmaceutical compositions of the present invention which are suitablefor vaginal administration also include pessaries, tampons, creams,gels, pastes, foams or spray formulations containing suchpharmaceutically-acceptable diluents or carriers as are known in the artto be appropriate.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, drops and inhalants. The active agent(s)/compound(s) may bemixed under sterile conditions with a suitablepharmaceutically-acceptable diluent or carrier. The ointments, pastes,creams and gels may contain excipients. Powders and sprays may containexcipients and propellants.

The pharmaceutical compositions of the present invention suitable forparenteral administrations may comprise one or more agent(s)/compound(s)in combination with one or more pharmaceutically-acceptable sterileisotonic aqueous or non-aqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsuitable antioxidants, buffers, solutes which render the formulationisotonic with the blood of the intended recipient, or suspending orthickening agents. Proper fluidity can be maintained, for example, bythe use of coating materials, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.These pharmaceutical compositions may also contain suitable adjuvants,such as wetting agents, emulsifying agents and dispersing agents. It mayalso be desirable to include isotonic agents. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption.

In some cases, in order to prolong the effect of a drug (e.g.,pharmaceutical formulation), it is desirable to slow its absorption fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material havingpoor water solubility.

The rate of absorption of the active agent/drug then depends upon itsrate of dissolution which, in turn, may depend upon crystal size andcrystalline form. Alternatively, delayed absorption of aparenterally-administered agent/drug may be accomplished by dissolvingor suspending the active agent/drug in an oil vehicle. Injectable depotforms may be made by forming microencapsule matrices of the activeingredient in biodegradable polymers. Depending on the ratio of theactive ingredient to polymer, and the nature of the particular polymeremployed, the rate of active ingredient release can be controlled. Depotinjectable formulations are also prepared by entrapping the drug inliposomes or microemulsions which are compatible with body tissue. Theinjectable materials can be sterilized for example, by filtrationthrough a bacterial-retaining filter.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquiddiluent or carrier, for example water for injection, immediately priorto use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the typedescribed above.

The present invention provides treatment of cancer which is refractoryor resistant to non-ERK MAPK pathway inhibitor therapy and disclosescombinations shown to enhance the effects of ERK inhibitors. Herein,applicants have also shown that the combination of different ERKinhibitors is likewise synergistic. Therefore, it is contemplated thatthe effects of the combinations described herein can be further improvedby the use of one or more additional ERK inhibitors. Accordingly, someembodiments of the present invention include one or more additional ERKinhibitors.

The present invention also provides a method of treating a subjecthaving an unresectable or metastatic BRAF600 mutation-positive melanomacomprising administering to the subject 600 mg BID of BVD-523 or apharmaceutically acceptable salt thereof.

In some embodiments of the invention, the mutation is a BRAFV600Emutation.

The present invention also provides a composition for treating a subjecthaving an unresectable or metastatic BRAF600 mutation-positive melanoma,the composition comprising 600 mg of BVD-523 or a pharmaceuticallyacceptable salt thereof and optionally a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The following examples are provided to further illustrate the methods ofthe present invention. These examples are illustrative only and are notintended to limit the scope of the invention in any way.

EXAMPLES Example 1 Materials and Methods

Cancer cell lines were maintained in cell culture under standard mediaand serum conditions. For dose escalation studies, A375 cells weresplit, grown to about 40-60% confluence, and then treated with theinitial dose of the specified drug. Table 4 shows a summary of drugtreatments that were escalated.

TABLE 4 Summary of Treatments Being Escalated Treatment Inhibitor 1Trametinib (MEKi) 2 Dabrafenib (BRAFi) 3 BVD-523 (ERKi) 4 Dabrafenib(BRAFi) + Trametinib (MEKi) 5 Dabrafenib (BRAFi) + BVD-523 (ERKi) 6Trametinib (MEKi) + BVD-523 (ERKi)

Single agent dose escalations were performed based on Little et al.,2011 and are outlined in FIG. 20. Cells were then allowed to grow until70-90% confluence and split. Split ratios were kept as “normal” aspossible and reasonably consistent between treatments (e.g. a minimum of50% of the normal split ratio of the parentals). Medium was refreshedevery 3-4 days. When cells again reached about 40-60% confluence, thedose was escalated. In the event that the 40-60% window was missed, thecells were split again and dosed once they reached 40-60% confluence.Again, medium was refreshed every 3-4 days. The process was repeated asrequired (FIG. 20).

For single agent treatments, starting concentrations and dose increaseswere conducted by starting with the approximate IC₅₀, escalating insmall increments or, gently, for the initial 4-5 doses, doubling thedose, increasing by the same increment for the next 4 doses, then movingto 1.5-fold increases in concentration for subsequent doses.

For combination treatments, starting concentrations and dose increaseswere conducted by starting with half of the approximate IC₅₀ of eachcompound (combination assay suggests this will result in about 40-70%inhibition range), escalating as per single agents (i.e. doing aninitial doubling and then increasing by the same increment for the next4 doses, then moving to 1.5-fold increases in concentration). Table 5shows the projected dose increases using these schemes.

TABLE 5 Projected Dose Increases-Month 1 Dab/Tram Dab/523 Tram/523 TramDab BVD-523 Dab Tram Dab 523 Tram 523 Dose (nM) (nM) (μM) (nM) (nM) (nM)(μM) (nM) (μM) 1 1 5 0.16 2.5 0.5 2.5 0.08 0.5 0.08 2 2 10 0.32 5 1 50.16 1 0.16 3 3 15 0.48 7.5 1.5 7.5 0.24 1.5 0.24 4 4 20 0.64 10 2 100.32 2 0.32 5 5 25 0.80 12.5 2.5 12.5 0.40 2.5 0.40 6 8 38 1.2 19 4 190.6 4 0.6 7 11 56 1.8 28 6 28 0.9 6 0.9 8 17 84 2.7 42 8 42 1.4 8 1.4 925 127 4.1 63 13 63 2.0 13 2.0 10 38 190 6.1 95 19 95 3.0 19 3.0 11 57285 9.1 142 28 142 4.6 28 4.6 12 85 427 13.7 214 43 214 6.8 43 6.8 13128 641 20.5 320 64 320 10.3 64 10.3 14 192 961 30.8 481 96 481 15.4 9615.4 15 288 1442 46.1 721 144 721 23.1 144 23.1 16 432 2162 69.2 1081216 1081 34.6 216 34.6 17 649 3244 103.8 1622 324 1622 51.9 324 51.9 18973 4865 155.7 2433 487 2433 77.8 487 77.8 19 1460 7298 233.5 3649 7303649 116.8 730 116.8 20 2189 10947 350.3 5474 1095 5474 175.2 1095 175.2

Clonal resistant cell populations were derived from resistant cell poolsby limiting dilution.

Proliferation assays were used to track changes in sensitivity to theescalated agent(s) at appropriate time intervals (e.g. each month,although the timing is dependent on adequate cell numbers beingavailable). For proliferation assays, cells were seeded in 96-wellplates at 3000 cells per well in drug-free DMEM medium containing 10%FBS and allowed to adhere overnight prior to addition of compound orvehicle control. Compounds were prepared from DMSO stocks to give afinal concentration range as shown in FIG. 2A-FIG. 2H. The final DMSOconcentration was constant at 0.1%. Test compounds were incubated withthe cells for 96 hours at 37° C. and 5% CO₂ in a humidified atmosphere.Alamar Blue 10% (v/v) was then added and incubated for 4 hours andfluorescent product was detected using a BMG FLUOstar plate reader. Theaverage media only background value was deducted and the data analyzedusing a 4-parameter logistic equation in GraphPad Prism. Paclitaxel wasused as a positive control.

Proliferation assays for month 1 were initiated at day 28 using cellsgrowing in the concentrations of each agent indicated in Table 6.

TABLE 6 Initial Concentrations of Drugs Used in Proliferation Assays -Month 1 Line Dab Tram BVD-523 Parental — — — Tram — 2 nM — Dab  15 nM —— BVD-523 — — 0.48 μM Tram + Dab   5 nM 1 nM — Dab + BVD-523 7.5 nM —0.24 μM Tram + BVD-523 — 1 nM 0.16 μM

Proliferation assays for month 2 were initiated at day 56 using cellsgrowing in the concentrations of each agent indicated in Table 7.

TABLE 7 Initial Concentrations of Drugs Used in Proliferation Assays -Month 2 Line Dab Tram BVD-523 Parental — — — Tram — 8 nM — Dab  127 nM —— BVD-523 — — 0.8 μM Tram + Dab   10 nM 2 nM — Dab + BVD-523 12.5 nM —0.4 μM Tram + BVD-523 — 2 nM 0.32 μM 

At the end of the 3 month escalation period, cultures were maintained atthe top concentration for 2 weeks prior to the final round ofproliferation assays and potential single cell cloning. As theproliferation assays/single cell cloning required actively proliferatingcells, for treatments where cells were proliferating very slowly at thetop concentration or that were only recently escalated, a backup culturewas also maintained at a lower concentration (Table 8). For the BVD-523treatment, where cells appeared to have almost completely stoppedgrowing and looked particularly fragile at the top concentration (1.8μM), cultures were maintained at a lower concentration for the 2 weekperiod.

TABLE 8 Details of Treatments Being Cultured at a Fixed Concentrationfor 2 Weeks Treatment Inhibitor Culture 1 Backup Culture 1 Tram 160 nM80 nM 2 Dab 3.2 μM — 3 BVD-523 1.2 μM 0.8 μM 4 Dab + Tram D: 160 nM D:80 nM T: 30 nM T: 16 nM 5 Dab + BVD-523 D: 42 nM D: 28 nM 523: 1.4 μM523: 0.9 μM 6 Tram + BVD-523 T: 4 nM T: 2.5 nM 523: 0.6 μM 523: 0.4 μM

Proliferation assays for month 3 used cells growing in theconcentrations of each agent indicated in Table 9.

TABLE 9 Initial Concentrations of Drugs Used in Proliferation Assays -Month 3 Line Dab Tram BVD-523 Parental — — — Tram — 160 nM — Dab 3.2 μM — — BVD-523 — — 1.2 μM Tram + Dab 80 nM  16 nM — Dab + BVD-523 28 nM —0.9 μM Tram + BVD-523 —  2.5 nM 0.4 μM

For combination studies, A375 cells (ATCC) were seeded into triplicate96-well plates at a cell density of 3000 cells/well in DMEM plus 10% FBSand allowed to adhere overnight prior to addition of test compound orvehicle control. Combinations were tested using a 10×8 dose matrix witha final DMSO concentration of 0.2%. A 96 hour assay incubation periodfollowed, with subsequent addition of Alamar Blue 10% (v/v) and 4 hoursincubation prior to reading on a fluorescent plate reader. After readingAlamar Blue, the medium/Alamar Blue mix was flicked off and 100 μl ofCellTiter-Glo/PBS (1:1) added and the plates processed as per themanufacturers instructions (Promega). Media only background values weresubtracted before the data was analysed. The Bliss additivity model wasthen applied.

In brief, predicted fractional inhibition values for combined inhibitionwere calculated using the equation C_(bliss)=A+B−(A×B) where A and B arethe fractional inhibitions obtained by drug A alone or drug B alone atspecific concentrations. C_(bliss) is the fractional inhibition thatwould be expected if the combination of the two drugs were exactlyadditive. C_(bliss) values are subtracted from the experimentallyobserved fractional inhibition values to give an ‘excess over Bliss’value. Excess over Bliss values greater than 0 indicate synergy, whereasvalues less than 0 indicate antagonism. Excess over Bliss values areplotted as heat maps±SD.

The single and combination data are also presented as dose-responsecurves generated in GraphPad Prism (plotted using % viability relativeto DMSO only treated controls).

For focused combination studies, the Alamar Blue viability assays wereperformed as described above for combination studies. Additionally,Caspase-Glo 3/7 assays were performed. In brief, HCT116 cells wereseeded in triplicate in white 96-well plates at a cell density of 5000cells/well in McCoy's 5A plus 10% FBS. A375 cells were seeded at adensity of 5000 cells/well in DMEM plus 10% FBS. Cells were allowed toadhere overnight prior to addition of test compound or vehicle control.The final concentration of DMSO was 0.2%, and 800 nM staurosporine wasincluded as a positive control. 24 and 48 hour assay incubation periodswere used. Then, Caspase-Glo® 3/7 50% (v/v) was added, plates were mixedfor 5 minutes on an orbital shaker and incubated for 1 hour at roomtemperature prior to reading on a luminescent plate reader. Media onlybackground values were subtracted before the data was analysed.

For Differential Scanning Fluorimetry, SYPRO orange (5,000× solution,Invitrogen) was diluted (1:1,000) in buffer solution (10 mM HEPES, 150mM NaCl, pH 7.5). HisX6 tagged proteins included inactive ERK2, activeERK2 (ppERK2), or p38a at a final concentration of 1 μM. The protein/dyesolution and compounds in 100% DMSO were added to wells (2% v/v finalDMSO concentration) to achieve the desired final concentrations, mixed,and placed into an RT-PCR instrument. Next, a melting curve was run from25-95° C. at a rate of 1° C. per minute and the melting temperature (Tm)was determined for each protein in the absence or presence of compounds.The change in Tm (ΔTm) in the presence of various drug concentrations ispresented.

For Ki determination of ERK1, activated ERK1 (10 nM) was incubated withvarious concentrations of the compounds in 2.5% (v/v) DMSO for 10minutes at 30° C. in 0.1 M HEPES buffer (pH 7.5), 10 mM MgCl₂, 2.5 mMphosphoenolpyruvate, 200 μM nicotinamide adenine dinucleotide (NADH),150 μg/mL pyruvate kinase, 50 μg/mL lactate dehydrogenase, and 200 μMErktide peptide. The reaction was initiated by the addition of 65 μM ofATP. Decreased absorbance rate (340 nm) was monitored and the IC₅₀ wasdetermined as a function of inhibitor concentration.

For Ki determination of ERK2, the inhibitory activity of BVD-523 againstERK2 was determined using a radiometric assay, with final concentrationof the components being 100 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mMdithiothreitol (DTT), 0.12 nM ERK2, 10 μM myelin basic protein (MBP),and 50 μM ³³P-γ-ATP. All reaction components, with the exception of ATPand MBP, were premixed and aliquoted (33 μL) into a 96-well plate. Astock solution of compound in DMSO was used to make up to 500-folddilutions; a 1.5-μL aliquot of DMSO or inhibitor in DMSO was added toeach well. The reaction was initiated by adding the substrates ³³P-γ-ATPand MBP (33 μL). After 20 minutes the reaction was quenched with 20%(w/v) tricholoracetic acid (TCA) (55 μL) containing 4 mM ATP,transferred to the GF/B filter plates, and washed 3 times with 5% (w/v)TCA). Following the addition of Ultimate Gold™ scintillant (50 μL), thesamples were counted in a Packard TopCount. From the activity versusconcentration titration curve, the Ki value was determined by fittingthe data to an equation for competitive tight binding inhibitionkinetics using Prism software, version 3.0.

For IC₅₀ determination of ERK2, activity was assayed by a standardcoupled-enzyme assay. The final concentrations were as follows: 0.1 MHEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 2.5 mM phosphoenolpyruvate, 200μM NADH, 50 μg/mL pyruvate kinase, 10 μg/mL lactate dehydrogenase, 65 μMATP, and 800 μM peptide (ATGPLSPGPFGRR). All of the reaction componentsexcept ATP were premixed with ERK and aliquoted into assay-plate wells.BVD-523 in DMSO was introduced into each well, keeping the concentrationof DMSO per well constant. BVD-523 concentrations spanned a 500-foldrange for each titration. The assay-plate was incubated at 30° C. for 10minutes in the plate reader compartment of the spectrophotometer(molecular devices) before initiating the reaction by adding ATP. Theabsorbance change at 340 nm was monitored as a function of time; theinitial slope corresponds to the rate of the reaction. The rate versusconcentration of the BVD-523 titration curve was fitted either to anequation for competitive tight-binding inhibition kinetics to determinea value for Ki or to a 3-parameter fit to determine the IC₅₀ using Prismsoftware, version 3.0.

For apoptosis assays, cells were plated at 2×10⁴ cells per well in a96-well plate and allowed to attach overnight or grow to 50% confluency.Cells were treated with a serial dilution of BVD-523 in media (finalvolume 200 μL, concentration ranges 4-0.25 μM) and incubated for 48hours in a 37° C. CO₂ incubator. Cells were washed with 100 μL of PBS,and 60 μL of radioimmunoprecipitation assay buffer was added (50 mMTris-HCl, pH 8.0, 150 mM NaCl, 1.0% [w/v] NP-40, 0.5% [w/v] sodiumdeoxycholate, 1% [w/v] SDS), then incubated for 10 minutes at 4° C. tolyse the cells. A 30-μL lysate aliquot was added to 100 μL of caspaseassay buffer (120 mM HEPES, 12 mM EDTA, 20 mM dithiothreitol, 12.5 μg/mLAC-DEVD-AMC caspase substrate) and incubated at RT from 4 hours toovernight. The plate was read in a fluorimeter (excitation wavelength360 nm, emission wavelength 460 mm). The remaining 30 μL of lysate wasanalyzed for total protein content using the BioRad Protein Assay Kit(sample-to-working reagent ratio of 1:8). Final normalized caspaseactivity was derived as fluorescence units per μg protein and convertedto a fold increase in caspase activity when compared with DMSO controls.

For measurement of antitumor activity in A375 xenografts, xenograftswere initiated with A375 cells maintained by serial subcutaneoustransplantation in female athymic nude mice. Each test mouse received anA375 tumor fragment (1 mm³) implanted subcutaneously in the right flank.Once tumors reached target size (80-120 mm³), animals were randomizedinto treatment and control groups, and drug treatment was initiated.

To evaluate BVD-523 monotherapy, BVD-523 in 1% (w/v)carboxymethylcellulose (CMC) was administered orally, per os (p.o.), BIDat doses of 5, 25, 50, 100, or 150 mg/kg. Oral temozolomide wasadministered as a positive reference compound at 75 or 175 mg/kg oncedaily (QD) for a total of five treatments (QD×5).

The efficacy of BVD-523 in combination with dabrafenib was evaluated inmice randomized into 9 groups of 15 and 1 group of 10 (Group 10).Dabrafenib was administered p.o. at 50 or 100 mg/kg QD and BVD-523 wasadministered p.o. at 50 or 100 mg/kg BID, alone and in combination,until study end; vehicle-treated and temozolomide-treated (150 mg/kgQD×5) control groups were also included. Combination dosing was stoppedon Day 20 to monitor for tumor regrowth. Animals were monitoredindividually and euthanized when each tumor reached an endpoint volumeof 2000 mm³, or the final day (Day 45), whichever came first, and mediantime to endpoint (TTE) calculated. The combination was also evaluated inan upstaged A375 model where larger tumors in the range 228-1008 mm³were evaluated. Here, mice were randomized into 1 group (Group 1) of 14and 4 groups (Groups 2-5) of 20. Dosing was initiated on Day 1 withdabrafenib plus BVD-523 (25 mg/kg dabrafenib+50 mg/kg BVD-523 or 50mg/kg dabrafenib+100 mg/kg BVD-523), with each agent given p.o. BIDuntil study end. The study included 50-mg/kg dabrafenib and 100-mg/kgBVD-523 monotherapy groups as well as a vehicle-treated control group.Tumors were measured twice weekly. Combination dosing was stopped on Day42 to monitor for tumor regrowth through study end (Day 60). Treatmentoutcome was determined from % TGD, defined as the percent increase inmedian TTE for treated versus control mice, with differences betweengroups analyzed via log rank survival analysis. For TGI analysis, % TGIvalues were calculated and reported for each treatment (T) group versusthe control (C) using the initial (i) and final (f) tumor measurementsbased on the following formula: % TGI=1−Tf−Ti/Cf−C. Mice were alsomonitored for CR and PR responses. Animals with a CR at the end of thestudy were additionally classified as TFS.

For measurement of BVD-523 activity in Colo205 xenografts, human Colo205cells were cultured in RPMI 1640 supplemented with 10% (v/v) fetalbovine serum (FBS), 100 units/mL penicillin, 100 μg/mL streptomycin(Invitrogen), and 2 mM L-glutamine. Cells were cultured for fewer thanfour passages prior to implantation. Female athymic nude mice (19-23 g)were injected subcutaneously with 2×10⁶ Colo205 cells into the rightdorsal axillary region on Day 0.

Mice with an approximate tumor volume of 200 mm³ were randomized into 6experimental groups. Vehicle control, 1% CMC (w/v), was prepared weekly.BVD-523 was suspended in 1% (w/v) CMC at the desired concentration andhomogenized on ice at 6,500 rpm for 50 minutes. BVD-523 suspensions wereprepared weekly and administered p.o. BID at total daily doses of 50,100, 150, and 200 mg/kg (n=12/group) on an 8- or 16-hour dosing schedulefor 13 days. The vehicle control (n=12) was administered using the samedosing regimen. CPT-11 was administered as a positive reference compound(n=12). Each 1 mL of CPT-11 injection contained 20 mg irinotecan, 45 mgsorbitol, and 0.9 mg lactic acid. CPT-11 was administered at 100mg/kg/day intraperitoneally every 4 days for 2 consecutive doses.

For measurement of ERK1/2 Isotope-Tagged Internal Standard (ITIS) MassSpectrometry in Colo205 Xenografts, frozen tumors were lysed in 10volumes of ice cold lysis buffer (10 mM TRIS-HCl, pH 8.0, 10 mM MgCl₂,1% (v/v) Triton X-100, Complete™ Protease Inhibitor Cocktail [Roche,cat. No. 1836170], Phosphatase Inhibitor Cocktail I [Sigma, cat. No.P-2850], Phosphatase Inhibitor Cocktail II [Sigma cat. No. 5726], andbenzonase [Novagen cat. No. 70664]). Lysates were clarified bycentrifugation (100,000×g for 60 minutes at 4° C.) and the supernatantsadjusted to 2 mg/mL with lysis buffer. ERK1 was immunoprecipitated usingagarose-coupled and pan-anti-ERK1 (Santa Cruz Biotechnology cat. No.sc-93ac) antibodies. Immunoprecipitated proteins were resolved bySDS-PAGE and stained with SYPRO Ruby (Invitrogen), and the ERK bandsexcised via razor. Gel slices were washed in 300 μL of 20 mM NH₄HCO₃,diced into small pieces, and placed in Page Eraser Tip (The Nest Groupcat no. SEM0007). Gel fragments were reduced and alkylated prior totrypsin digestion. Tryptic fragments were isolated in 75 μL of 50% (v/v)Acetonitrile, 0.2% (v/v) trifluoroacetic acid and the resulting sampleconcentrated to 0-10 μL in a SpeedVac.

For ITIS analysis, digested samples were spiked with heavy-atom labeledpeptide standards and fractional phosphorylation was quantified bycoupled liquid chromatography-tandem mass spectrometry (MS).Nanocapillary chromatography was performed using a Rheos 2000 binarypump from Flux Instruments delivering nanoscale flow after 1:750splitting, an LC Packings Inertsil nano-precolumn (C18, 5 mm, 100 Å, 30mm ID×1 mm), and a New Objective PicoFrit AQUASIL resolving column (C18,5 mm, 75/15 mm ID×10 cm), which also served as an electrosprayionization (ESI) emitter. An Applied Biosystem API 3000 massspectrometer coupled with a nano-ESI source was used for MS analysis. Anin-house-made gas nozzle connected to a nebulizing gas source was usedto help steady nano-flow spray. Data were acquired in a multiplereaction monitoring (MRM) mode: nebulizing gas at 3; curtain gas at 7;collision gas at 5; ion spray voltage at 2150 volts, exit potential at10 volts; Q1/Q3 resolution Low/Unit; and dwell time of 65 msec for allMRM channels. All raw MS data were processed using a combination of theAnalyst software suite from Applied Biosystem and custom tools.

For assessment of drug sensitivity in cell-line models of acquiredresistance, drug sensitivity of dose-escalated A375 cells and isogenicRKO cells was assessed in 96-hour proliferation assays. RKO isogeniccells (McCoy's 5A containing 10% [v/v] FBS) or dose-escalated A375 cells(DMEM containing 10% FBS were seeded into 96-well plates and allowed toadhere overnight prior to addition of compound or vehicle control. Notethat the dose-escalated A375 cells were seeded in the absence ofinhibitor. Compounds were prepared from 0.1% (v/v) DMSO stocks to give afinal concentration as indicated. Test compounds were incubated with thecells for 96 hours at 37° C. in a 5% CO₂ humidified atmosphere. For theRKO cells, CellTiter-Glo® reagent (Promega) was added according tomanufacturer's instructions and luminescence detected using a BMGFLUOstar plate reader. For the A375 assays Alamar blue (ThermoFisher)10% (v/v) was added and incubated for 4 h, and fluorescent product wasthen detected using a BMG FLUOstar. The average media only backgroundvalue was deducted and the data analyzed using a 4-parameter logisticequation in GraphPad Prism.

IC₅₀ Determination of ERK1 was measured in a final reaction volume of 25μL. ERK1 (human) (5-10 mU) was incubated with 25 mM Tris (pH 7.5), 0.02mM ethyleneglycoltetracetic acid, 250 μM peptide, 10 mM Mg acetate, andγ-³³P-ATP (specific activity approximately 500 cpm/pmol, concentrationas required). Adding Mg ATP initiated the reaction. After incubation for40 minutes at room temperature (RT), the reaction was stopped by adding5 μL of a 3% (w/v) phosphoric acid solution. Then, 10 μL of the reactionwas spotted onto a P30 filtermat, and washed 3 times for 5 minutes in 75mM of phosphoric acid then once in methanol before drying andscintillation counting.

RKO MEK1 Q56P Isogenic cells were produced by Horizon Discovery(Cambridge, UK; #HD 106-019) using a recombinant AAV-mediated genetargeting strategy. Briefly, rAAV virus was generated followingtransfection of the appropriate targeting vector and helper vectors inHEK293T cells, purified using an AAV purification kit (Virapur, SanDiego, USA) and titrated using qPCR. Parental homozygous RKO cells(homozygous wild type for MEK1) were then infected with rAAV virus andclones that had integrated the selection cassette were identified byG418 selection and expanded. Correctly targeted clones that wereheterozygous for knock-in of the MEK1 Q56P point mutation into a singleallele were identified by PCR and sequencing.

Isogenic SW48 cell lines heterozygous for knock-in of mutant KRAS (DeRoock et al 2010, JAMA, 304, 1812-1820) were obtained from HorizonDiscovery (Catalogue numbers; HD 103-002, HD 103-006 HD 103-007, HD103-009, HD 103-010, HD 103-011, HD 103-013). For proliferation assay,cells were seeded into 96-well plates in McCoy's 5A medium supplementedwith 10% FBS and allowed to adhere overnight prior to addition ofcompound or vehicle control. Test compounds were incubated with thecells for 96 hours at 37° C. in a 5% CO₂ atmosphere. Viability was thenassessed using Alamar blue.

The proprietary KinaseProfiler assay was conducted at Upstate Discoveryand employed radiometric detection similar to that employed by Davies etal, was used to profile the selectivity of BVD-523 against a panel of 70kinases.

A drug sensitivity analysis was carried out as part of The Genomics ofDrug Sensitivity in Cancer Project using high-throughput screening, aspreviously described (Yang et al. 2013).

For Western blot analysis, A375 cells were seeded onto 10 cm dishes inDulbecco's Modified Eagle's Medium plus 10% (v/v) FBS. Cells wereallowed to adhere overnight prior to the addition of test compound orvehicle. For experiments with RKO cells, these cells were seeded in6-well plates or 10 cm dishes with McCoy's 5A+10% (v/v) FBS. Cells werethen treated at the desired concentration and duration. Cells wereharvested by trypsinization, pelleted, and snap frozen. Lysates wereprepared with RIPA buffer supplemented with protease and phosphataseinhibitor cocktails (Roche), clarified by centrifugation at 11,000 rpmfor 10 minutes, and quantitated by bicinchoninic acid assay. Sampleswere resolved by SDS-PAGE, blotted onto polyvinylidene difluoridemembranes, and probed using antibodies (i.e., pRB [Ser780], cat. no.9307; CCND1, cat. no. ab6152; BCL-xL, cat. no. 2762; PARP, cat. no.9542; DUSP6, cat. no. 3058S) directed to the indicated targets.

For Reverse Phase Protein Analysis (RPPA), A375, MIAPaCa-2, HCT116,Colo205, HT-29, and AN3Ca cells (ATCC) were plated at 80% confluence,allowed to recover overnight (MIAPaCa-2 cells were plated at 30%confluence and allowed to recover for 3 days), then treated with 10 μMof each compound (i.e., BVD-523, SCH722984, GDC-0994, or Vx-11e) for 6hours at 37° C. Control wells were treated with DMSO at 0.1% (v/v) for 6hours prior to cell lysate generation. Samples were then analyzed usingreverse-phase protein microarray technology (Theranostics Health).

For analysis of pERK IHC in Colo205 xenografts, xenograft tumors wereprocessed overnight in 70% through 100% graded ethanols, cleared in twochanges of xylene, infiltrated with paraffin, and embedded into paraffinblocks. Then, 5-μm sections were cut and placed onto positively chargedglass slides and baked for at least 30 minutes, but not longer than 1hour, at 60° C. A single section from each animal and dose group wasprobed with anti-phospho p42/p44 MAPK antibody (pERK [1:100], CST; Catno. 9101; Lot no. 16), counterstained with hematoxylin, and thenanalyzed microscopically using a Zeiss Axioplan 2 microscope. An isotypecontrol (rabbit, Zymed laboratories, catalog no. 08-6199, lot no.40186458) was run as a negative control.

For FACS analysis, cells were scraped and pelleted at 1,500 rpm for 5minutes, then re-suspended in 1 mL of buffer and frozen at −70° C. Thefrozen cells were thawed and centrifuged again, followed by 10 minutesof re-suspension in 0.25 mL of Buffer A (trypsin in sperminetetrahydrochloride detergent buffer) to disaggregate cell clumps anddigest cell membranes and cytoskeletons. Buffer B (trypsin inhibitor andRibonuclease I in buffer, 0.2 mL) was added for 10 minutes in the dark.The resulting DNA-stained nuclei were filtered and analyzed by FACS. Thehistograms were analyzed to establish the proportion of cells in the G1,S, and G2/M phases of the cell cycle based on the presence of n and 2nDNA (or higher) content.

For measurement of in vitro combination activity, five thousand G-361cells were seeded into triplicate 96-well plates containing McCoy's 5Awith 10% (v/v) FBS and allowed to adhere overnight. Thevemurafenib/BVD-523 combination was tested using a 10×8 dose matrix.Compounds were incubated with the cells for 72 hours at 37° C. in a 5%CO₂ humidified atmosphere. CellTiter-Glo reagent was added according tomanufacturer's instructions and luminescence detected using a MBGFLUOstar plate reader. The interactions across the dose matrix weredetermined by the Loewe Additivity and Bliss independence models usingHorizon's Chalice Combination Analysis Software.

For generating compound resistance in vitro by dose escalation, A375parental cells (ATCC CRL-1619) were grown to ˜40-60% confluence inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%heat-inactivated FBS and penicillin/streptomycin, then treated withinitial doses of BVD-523, trametinib, or dabrafenib either alone or incombination at or slightly below each compound's IC₅₀; for combinationstudies, initial dosing was half of each compound's IC₅₀. Cells wereallowed to grow until ˜70-90% confluence and split; medium was refreshedevery 3-4 days. When cells again reached ˜40-60% confluence, the dosewas escalated by the same increment (equivalent to the startingconcentration) then moved to 1.5-fold increases in concentrationfollowed by a further move to 2-fold increases if the cells continued toadapt rapidly (e.g., the first six doses of the dabrafenib escalationwere: 5, 10, 15, 20, 25, and 37.5 nM). This process was repeated asrequired.

Cell viability assays for FIG. 30A were performed by a Resazurin (AlamarBlue) metabolic assay after 5 days in drug in full serum under highglucose conditions. Cells were seeded in 384-well microplates at˜15%-50% confluence in medium with 10% FBS and penicillin/streptavidinplus high glucose (18-25 mM). The optimal cell number for each cell linewas determined to optimize growth during drugging. For adherent celllines, after overnight incubation cells were treated with 9concentrations of each compound (2-fold dilutions series) using liquidhandling robotics, and returned to the incubator for assay at a 96-htime point. For suspension cell lines, cells were treated with compoundimmediately after plating and returned to the incubator for a 96-h timepoint. Cells were then stained with 55 μg/ml Resazurin (Sigma) preparedin glutathione-free media for 4 hours. Quantitation of fluorescentsignal intensity was performed using a fluorescent plate reader atexcitation and emission wavelengths of 535/595 nm for Resazurin. Allscreening plates were subjected to stringent quality control measures.Effects on cell viability were measured and a curve-fitting algorithmwas applied to the raw dataset to derive a multi-parameter descriptionof drug response, including the half maximal inhibitory concentration(IC₅₀). IC₅₀ is expressed in natural log of the IC₅₀ in μM (LN_IC₅₀; EXPreturns IC₅₀ in μM). Extrapolation of the IC₅₀ was allowed for where ityielded very high values. If desired the data was restricted to thetested concentration range by capping IC₅₀ values at the maximum testedconcentration (and the minimum tested concentration for low values).

For efficacy testing of BVD-523 in a patient-derived xenograft (AT052C)representing melanoma from a BRAF^(V600E) patient that had becomeclinically refractory to vemurafenib. Tumor fragments were harvestedfrom host animals and implanted into immune-deficient mice. The studywas initiated at a mean tumor volume of approximately 170 mm³, at whichpoint the animals were randomized into four groups including a control(1% [v/v] CMC p.o., BID×31) and three treatment groups (BVD-523 [100mg/kg], dabrafenib [50 mg/kg], or BVD-523/dabrafenib [100/50 mg/kg],n=10/group); All treatment drugs were administered p.o. on a BID×31schedule.

For IC₅₀ determination for the inhibition of PMA-stimulated RSK1phosphorylation by BVD-523 in human whole blood samples, IC₅₀ values forthe inhibition of PMA stimulated RSK1 phosphorylation by BVD-523 weredetermined for 10 healthy donors (aged 22-61 years) using an 8-pointconcentration curve ranging from 10 μM to 5 nM of BVD-523. Controlsconsisted of 3 unstimulated samples and 3 PMA-stimulated samples foreach donor. Both phosphor-RSK (pRSK) and total RSK levels weredetermined and data were calculated using pRSK/RSK levels for eachsample.

Thirty milliliters of blood was drawn from each donor into sodiumheparin vacutainers. One mL of whole blood was added to each oftwenty-two 2-mL microtubes per donor. The microtubes tubes were labeledwith the donor number (1 through 10) and the subsequent treatmentdesignation: “A” for PMA stimulation only (maximum), “B” forBVD-523-containing samples that received PMA stimulation; and “C” forthe unstimulated samples (minimum). Dimethyl sulfoxide (DMSO) was addedto all tubes in groups A and C to a final concentration of 0.1%. Sampleswere then rocked gently at room temperature.

BVD-523 (10 mM in 100% DMSO) was serially diluted with 3-fold dilutionsinto 100% DMSO. These serially diluted BVD-523 samples in 100% DMSO werethen diluted 10-fold in Dulbecco's Modified Eagle Medium containing 10%fetal bovine serum and penicillin/streptomycin/glutamine, and 10 μL ofeach of these working solutions was added per mL of blood for eachdesignated BVD-523 concentration. Each concentration of BVD-523 was runin duplicate, two 1-mL blood samples each, yielding 16 total samples forthe full 8-point concentration curve. Samples were then rocked gently atroom temperature for a minimum of 2 hours but not longer than 3 hours.

Human whole blood samples in groups A and B for all donors werestimulated with PMA at a final concentration of 100 nM for 20 minutes atroom temperature. Samples in group C were not treated with PMA but wererocked and handled as all other samples.

Upon completion of PMA treatment for each sample, peripheral bloodmononuclear cells were isolated from the human whole blood. One mL ofblood from each sample was gently layered onto 0.75 mL ofroom-temperature Histopaque 1077 in a 2-mL microcentrifuge tube. Thesamples were centrifuged for 2 minutes at 16,000×g in an Eppendorfmicrocentrifuge. The interface and upper layers were removed and addedto tubes containing 1 mL of cold Dulbecco's phosphate-buffered saline(DPBS). These samples were then centrifuged for 30 seconds at 16,000×gto pellet the cells. The buffer supernatant was removed by aspirationand the pellets were re-suspended in 1 mL of cold DPBS. The pellets fromeach sample were then re-pelleted as above. The buffer was removed byaspiration and the pellets were lysed as indicated below.

Complete lysis buffer consisted of Meso Scale Discovery Tris lysisbuffer, 1× Halt Protease inhibitor cocktail, 1× Phosphatase inhibitorcocktail 2, 1× Phosphatase inhibitor cocktail 3, 2 mMphenylmethanesulfonyl fluoride, and 0.1% sodium dodecyl sulfate. Lysisbuffer was kept on ice and made fresh for each sample group. Final cellpellets were lysed by the addition of 120 μL of complete lysis buffer.Samples were vortexed until the cell pellet disappeared and then flashfrozen on dry ice. Samples were stored at −20° C. prior to measurementof pRSK and total RSK by ELISA.

For the pRSK ELISA (PathScan), thawed lysates were combined 1:1 withsample diluent (provided in ELISA kit): 120 μL of lysate added to 120 μLof sample diluent in a round bottom 96-well plate. This combination wasthen transferred to the pRSK microwells at 100 μL per well. For thetotal RSK ELISA (PathScan), 20 μL of the lysate already diluted 1:1 insample diluent was further diluted in 200 μL of sample diluent in around bottom 96-well plate. This combination was then transferred to thetotal RSK microwells at 100 μL per well. The plates were sealed with aplate seal and incubated 16 to 18 hours at 4° C., a time that was shownto yield the best detection of the target protein. Both ELISAs weredeveloped according to the kit instructions.

Patients aged ≥18 years were eligible for participation if they hadnoncurable, histologically confirmed metastatic or advanced stagemalignant tumors; an ECOG performance status of 0 or 1; adequate renal,hepatic, bone marrow, and cardiac function; and a life expectancy ≥3months. Patients may have received up to 2 prior lines of chemotherapyfor their metastatic disease. Exclusion criteria were known uncontrolledbrain metastases; gastrointestinal conditions which could impairabsorption of study medication; history or current evidence/risk ofretinal vein occlusion or central serous retinopathy; and concurrenttherapy with drugs known to be strong inhibitors of CYP1A2, CYP2D6, andCYP3A4 or strong inducers of CYP3A4. All participants provided informedconsent prior to initiation of any study procedures.

Patients that received at least one dose of BVD-523 were included in theanalysis useing SAS (version 9.3) software. The data cutoff was Dec. 1,2016. This study is registered with ClinicalTrials.gov, numberNCT01781429.

The present invention presents data from an open-label, multicenterphase I study to assess the safety, pharmacokinetics, andpharmacodynamics of escalating doses of BVD-523 in patients withadvanced malignancies. The dosing regimen combined both acceleratedtitration and standard cohort 3+3 dose escalation schema, which wereused jointly to identify the MTD and RP2D of BVD-523 in patients withadvanced solid tumors. One to 6 patients per treatment cohort wereassigned to receive sequentially higher oral doses of BVD-523 on a BIDschedule (12-hour intervals) in 21-day cycles, starting at a dose of 10mg BID. BVD-523 was administered BID continuously in 21-day cycles atthe following doses: 10 mg (n=1); 20 mg (n=1); 40 mg (n=1); 75 mg (n=1);150 mg (n=1); 300 mg (n=4); 600 mg (n=7); 750 mg (n=4); and 900 mg(n=7).

Patients received BID oral doses until disease progression, unacceptabletoxicity, or a clinical observation satisfying another withdrawalcriterion. Dose escalations occurred in up to 100% increments insingle-patient cohorts until 1 patient experienced a ≥Grade 2 toxicity(excluding alopecia or diarrhea). Cohorts were then expanded to at least3 patients each and subsequent dose-escalation increments were reducedfrom up to 100% to a maximum of 50%. When at least 1 patient in a3-patient cohort experienced a DLT, up to 3 additional patients weretreated at this dose level. When more than 1 DLT occurred in patients,this dose level was defined as the nontolerated dose and dose escalationwas stopped. Intrapatient dose escalation was allowed, provided thepatients receiving the highest current dose had been observed for atleast 3 weeks and dose-limiting side effects were reported in fewer than2 of 6 patients assigned to a given dose. Patients experiencing DLTs orunacceptable toxicity had their treatment interrupted until the toxicityreturned to ≤Grade 1. Resumption of BVD-523 treatment was then initiatedat the next lower dose level tested or at a 20% to 30% dose decrease,aligning with capsule dosage.

The primary objective of the phase I study was to define the safety andtolerability of BVD-523 by determining the dose-limiting toxicities, theMTD, and the RP2D. The secondary objectives included the determinationof the pharmacokinetic profile of BVD-523 in patients with advancedmalignancies and the investigation of any preliminary clinical effectson tumor response, as assessed by physical or radiologic exam usingRECIST v1.1. The exploratory objectives included evaluation ofpharmacodynamic marker (biomarker) measures and investigation ofpreliminary clinical effects on tumor response assessed by ¹⁸F-FDG-PETas indicated.

For determination of MTD, DLT, and RP2D, MTD was defined as the highestdose cohort at which ≤33% of patients experienced BVD-523-related DLTsin the first 21 days of treatment. DLT was defined as a BVD-relatedtoxicity in the first 21 days of treatment that resulted in ≥Grade 4hematologic toxicity for >1 day; Grade 3 hematologic toxicity withcomplications (e.g., thrombocytopenia with bleeding); ≥Grade 3nonhematologic toxicity, except untreated nausea, vomiting,constipation, pain, and rash (these become DLTs if the AE persisteddespite adequate treatment); or a treatment interruption exceeding 3days in Cycle 1 (or the inability to being in Cycle 2 for >7 days) dueto BVD-523-related toxicity.

The RP2D could be as high as the MTD and was determined in discussionwith the clinical investigators, the medical monitor, and the sponsor.Observations related to pharmacokinetics, pharmacodynamics, and anycumulative toxicity observed after multiple cycles were included in therationale supporting the RP2D.

With regard to safety assessments, AEs were defined as any untowardmedical occurrence in a patient who was administered a medicinal productthat does not necessarily have a causal relationship with BVD-523, andwas coded using the MedDRA coding dictionary. An SAE was any untowardmedical occurrence that occurred at any dose that resulted in death, waslife-threatening, required inpatient hospitalization or prolongation ofexisting hospitalization, or resulted in persistent or significantdisability/incapacity or a congenital anomaly/birth defect. The severityof AEs were graded according to the National Cancer Institute CommonTerminology Criteria for Adverse Events, Grading Scale, version 4.

Safety evaluations were conducted at baseline, on Days 8, 15, 22, 29,36, and 43, and, in patients who continued treatment, every 3 weeks orif clinically indicated thereafter. Each evaluation included a physicalexamination and clinical laboratory studies. Electrocardiograms wererepeated if clinically significant and at the discretion of theinvestigator. The investigators made judgments regarding whether or notAEs were related to study drug and followed up until resolution orstabilization, or the AE was judged to be no longer clinicallysignificant.

For pharmacokinetic analysis, the pharmacokinetic population consistedof patients who received at least one dose of BVD-523 and had evaluablepharmacokinetic data for plasma and/or urine. Blood samples werecollected prior to dosing, and then at 0.5 (±5 min), 1 (±5 min), 2 (±10min), 4 (±10 min), 6 (±10 min), 8 (±10 min), and 12 (±2 hr) hours on Day1 (Visit 2; baseline/initiation of treatment) and Day 15 (Visit 4; atsteady state) after the morning dose. On Day 22, prior to doseadministration, a final blood sample was collected for pharmacokineticanalyses. Urine samples were collected predose and at the 1- to 6-hourand 6- to 12-±2-hour intervals postdose on Days 1 and 15. Plasma andurine samples were analyzed for BVD-523 and metabolites using validatedLC/MS/MS methods. Standard pharmacokinetic parameters were obtainedusing Phoenix WinNonlin (Pharsight) with a noncompartmental method.Relationship between dose and exposure was calculated using standardleast-squares regression analysis.

For pharmacodynamic confirmation of target inhibition by BVD-523,targeted ERK inhibition by BVD-523 was determined by examining pRSK as atarget biomarker in human whole blood samples obtained from patientswith advanced solid tumors (N=27) who had received different doses ofBVD-523 (10-900 mg BID) during the phase I study. The activity ofBVD-523 from 4 timepoints (baseline predose, baseline 4 hours postdose,Day 15 predose, and Day 15 4 hours postdose) was expressed as a percentactivity (pRSK) of PMA-stimulated blood incubated with BVD-523.

For measurement of antitumor response, tumor measurements based onphysical examination occurred at baseline and on the first day of eachtreatment cycle. CT and other assessments were made every 2 to 3 cycles.Findings were assessed in accordance with RECIST v1.1: CR was defined asdisappearance of all target lesions; PR was defined as a ≥30% decreasein the sum of the longest diameters of target lesions, taking baselinemeasurements as reference; stable disease was defined as being ofneither sufficient shrinkage to qualify for PR nor sufficient increaseto qualify for progressive disease, taking as reference the baselinemeasurement. Metabolic response was assessed by visualizing tumor uptakeof ¹⁸F-glucose via ¹⁸F-FDG-PET scanning prior to receiving the firstdose of BVD-523 and at Day 15 (Visit 4).

Example 2 Dose Escalation and Proliferation Assays—Month 1

Dose Escalation Progress—Month 1

A375 cells were dose escalated using BVD-523, dabrafenib, and trametinibeither as single agents or in combination. Doses were increased in smallincrements during the first month. Other than a marked reduction ingrowth rate, cells generally tolerated the escalations well and thedoses were planned to be more aggressively escalated using largerincrements in month 2. FIG. 1A-FIG. 1C show month 1 progress for thedose escalation studies.

Proliferation Assay Results—Month 1

Proliferation assays were performed to assess the response of theescalated cells lines vs. parental cell line, to BVD-523, dabrafenib,and trametinib treatments.

FIG. 2A-FIG. 2H show normalized and raw proliferation assay results frommonth 1 of the studies. Note that differences in max signals in DMSOcontrols between different treatments (FIG. 2D, FIG. 2F, and FIG. 2H)suggest differential growth rates between treatments. These differencesmay influence the responses of lines to inhibitors in the proliferationassays.

Table 10 shows IC₅₀ data for month 1 of the studies.

TABLE 10 IC₅₀ Data-Month 1 Cell Line, Relative IC₅₀ (nM) BVD- Dab/ Dab/Tram/ Compound Par* Tram Dab 523 Tram 523 523 Dabrafenib 6 29 about 8 5868 11 161 Trametinib 0.5 2.2 2.5 0.7 3.9 3.1 2.5 BVD-523 189 335 350 268300 412 263 Paclitaxel 2.2 3.0 3.3 3.4 3.5 3.4 3.4 *Par = Parental cellline

There were early hints that cells grown in the presence of escalatingdoses of dabrafenib or trametinib, either as single agents or incombinations, were exhibiting decreased responses to these two agents inproliferation assays.

In the early stages of month 2, the growth rate of cells in thedabrafenib only treatment notably increased relative to the early stagesof month 1. This enabled an increased rate of progression and suggestedthat resistance was becoming apparent.

Example 3 Dose Escalation and Proliferation Assays—Month 2

Dose Escalation Progress—Month 2

The second month of studies saw most treatments move into a phase wheredoses were increased in greater increments (1.5-fold) compared to theinitial gentle escalation phase. The single agent escalation ofdabrafenib and trametinib was quickest, with cells growing inconcentrations equivalent to 100× parental cell IC₅₀ (FIG. 3A and FIG.3B). The single agent escalation of BVD-523 progressed more slowlycompared to dabrafenib and trametinib (FIG. 3C). See FIG. 3D for acomparison of the single agent escalations. BVD-523 escalated cells hada more “fragile” appearance and there was a greater number of floatingcells compared to the dabrafenib and trametinib escalated populations.

The combined agent escalations progressed more slowly than the singleagent treatments. The BVD-523/trametinib combination was particularlyeffective in preventing cells from progressing.

Proliferation Assay Results—Month 2

Proliferation assays on single agent escalated dabrafenib and trametinibcell populations revealed modest shifts in the dose response curves,suggesting that an additional period of escalation would be beneficialto further enrich for resistant cells. Interestingly, in theproliferations assay, there was evidence to suggest that cells exposedto BVD-523 grew less well upon inhibitor withdrawal, perhaps indicatinga level of addiction.

FIG. 4A-FIG. 4H show normalized and raw proliferation assay results frommonth 2 of the studies. Note that differences in max signals in DMSOcontrols between different treatments (FIG. 4D, FIG. 4F, and FIG. 4H)suggest differential growth rates between treatments. These differencesmay influence the responses of lines to inhibitors in the proliferationassays.

FIG. 5A-FIG. 5H show normalized and raw proliferation assay results frommonth 2 of the studies with a focus on parental and BVD-523 line dataonly.

Table 11 shows IC₅₀ data for month 2 of the studies. Relative IC₅₀s weredetermined from 4-parameter curve fits in Prism.

TABLE 11 IC₅₀ Data-Month 2 Cell Line, Relative IC₅₀ (nM) BVD- Dab/ Dab/Tram/ Compound Par* Tram Dab 523 Tram 523 523 Dabrafenib 4.1 6.2 11.5697 256 218 68 Trametinib 0.4 0.7 1.1 24.3 12.6 6.2 4.6 BVD-523 187 252284 1706 561 678 435 Paclitaxel 3.7 8.9 1.9 6.5 4.7 4.2 8.9 *Par =Parental cell line

Example 4 Dose Escalation and Proliferation Assays—Month 3

Dose Escalation Progress—Month 3

FIG. 6A-FIG. 6C show single and combination agent escalation for month 3of the studies. FIG. 6D shows a comparison of single agent escalations.

Proliferation Assay Results—Month 3

FIG. 7 shows an assessment of growth during the proliferation assay inDMSO control wells. FIG. 8A-FIG. 8D show results from month 3 of thestudies. FIG. 9A-FIG. 9D show results from month 3 of the studies with afocus on single treatment cell lines.

Table 12 shows IC₅₀ data for month 3 of the studies. Relative IC₅₀s weredetermined from 4-parameter curve fits in Prism. IC₅₀ values were notdetermined for the cell line escalated with trametinib due to a lack ofgrowth during the assay (ND: not done).

TABLE 12 IC₅₀ Data-Month 3 Cell Line, Relative IC₅₀ (nM) BVD- Dab/ Dab/Tram/ Compound Par* Tram Dab 523 Tram 523 523 Dabrafenib 2.1 ND 2.5 18.417.9 337 73 Trametinib 0.2 ND 0.4 1.7 2.7 90 11.2 BVD-523 129 ND 198 433323 1151 296 Paclitaxel 1.9 ND 1.9 6.5 4.7 4.2 8.9 *Par = Parental cellline

FIG. 19 shows single and combination agent escalation for month 3 of thestudies. Cell line variants were obtained that could grow in thepresence of dabrafenib or trametinib at concentrations greater than 100times the IC₅₀ of these agents in parental A375 cell. In comparison,cell lines resistant to BVD-523 could only be maintained in less than10× of parental IC₅₀ concentration. Sensitivity testing suggesteddabrafenib and trametinib-resistant cell lines remained relativelysensitive to BVD-523; the increased IC₅₀ “shift” for BVD-523 inresistant cell lines was more modest than those corresponding IC₅₀increases following dabrafenib or trametinib treatment. Likewise,compared to dabrafenib or trametinib treatment, more complete inhibitionof cell growth was observed when resistant cell lines were treated withBVD-523 at concentrations 10-fold above its IC₅₀ in the parental A375line. In total, patterns of resistance and cross-sensitivity suggestBVD-523 may remain effective in settings of acquired resistance.

Example 5 Combination Study Results

As expected, A375 cells, which carry a BRAF (V600E) mutation, weresensitive to dabrafenib. Single agent IC₅₀ values calculated usingAlamar Blue (FIG. 10A-FIG. 10E, FIG. 12A-FIG. 12E, and FIG. 14A-FIG.14E) were generally slightly lower for Dabrafenib and BVD-523 comparedto those derived using CellTiter-Glo (FIG. 11A-FIG. 11E, FIG. 13A-FIG.13E, and FIG. 15A-FIG. 15E). Published IC₅₀ values for Dabrafenib andTrametinib in a 72 hour CellTiter-Glo assay were 28±16 nM and 5±3 nMrespectively (Greger et al., 2012; King et al., 2013)—the single agentresults reported here are consistent with these values. There was someevidence for a window of synergy in all treatments. Variation betweentriplicates was low, however, there was some evidence of edge effectsthat likely explains the apparent enhanced growth observed in sometreatments versus the no drug control (e.g. particularly apparent in theTrametinib/BVD-523 combination). This makes the interpretation of theBliss analysis more challenging as in some treatments it may haveresulted in the artefactual enhancement in the level of synergy.

The combination assays were repeated for A375 cells. Single agentBVD-523, Trametinib and Dabrafenib potencies were consistent with thosereported in the previous studies disclosed herein.

In sum, taken together the data show that MEK and BRAF resistant cellscould be overcome by treatment with the ERK inhibitor, BVD-523.

Example 6 BVD-523 Altered Markers of MAPK Kinase Activity and EffectorFunction

For Western blot studies, HCT116 cells (5×10⁶) were seeded into 10 cmdishes in McCoy's 5A plus 10% FBS. A375 cells (2.5×10⁶) were seeded into10 cm dishes in DMEM plus 10% FBS. Cells were allowed to adhereovernight prior to addition of the indicated amount of test compound(BVD-523) or vehicle control. Cells were treated for either 4 or 24hours before isolation of whole-cell protein lysates, as specifiedbelow. Cells were harvested by trypsinisation, pelleted and snap frozen.Lysates were prepared with RIPA (Radio-Immunoprecipitation Assay)buffer, clarified by centrifugation and quantitated by bicinchoninicacid assay (BCA) assay. 20-50 μg of protein was resolved by SDS-PAGEelectrophoresis, blotted onto PVDF membrane and probed using theantibodies detailed in Table 13 (for the 4-hour treatment) and Table 14(for the 24-hour treatment) below.

TABLE 13 Antibody Details Incubation/ Size Block Antigen (kDa) SupplierCat No Dilution Conditions Secondary pRSK1/2 90 Cell 9335 1:1000 o/n 4°C. 5% anti-rabbit p5380 Signaling BSA pRSK1/2 90 Cell 11989 1:2000 o/n4° C. 5% anti-rabbit pS380 Signaling BSA pRSK- 90 Millipore 04-4191:40000 o/n 4° C. 5% anti-rabbit T359/5363 BSA Total 90 Cell 9333 1:1000o/n 4° C. 5% anti-rabbit RSK Signaling BSA pErk 1/2 42/44 Cell 9106S1:500 o/n 4° C. 5% anti-mouse Signaling milk Total 42/44 Cell 91021:2000 o/n 4° C. 5% anti-rabbit ERK Signaling milk pMEK1/2 45 Cell 91541:1000 o/n 4° C. 5% anti-rabbit Signaling BSA Total 45 Cell 9126 1:1000o/n 4° C. 5% anti-rabbit MEK Signaling BSA pS6-pS235 32 Cell 2211S1:3000 o/n 4° C. 5% anti-rabbit Signaling milk Total S6 32 Cell 22171:2000 o/n 4° C. 5% anti-rabbit Signaling milk DUSP6 48 Cell 3058S1:1000 o/n 4° C. 5% anti-rabbit Signaling BSA Total 73 BD Bio- 6101521:2000 o/n 4° C. 5% anti-mouse CRAF sciences milk pCRAF- 73 Cell 94271:1000 o/n 4° C. 5% anti-rabbit Ser338 Signaling BSA pRB 105 Cell 93071:2000 o/n 4° C. 5% anti-rabbit (Ser780) Signaling BSA β-Actin 42 SigmaA5441 1:500,000 o/n 4° C. 5% anti-mouse milk

TABLE 14 Antibody details Incubation/ Size Block Antigen (kDa) SupplierCat No Dilution Conditions Secondary pRB 105 Cell 9307 1:2000 o/n 4° C.5% anti-rabbit (Ser780) Signaling BSA CCND1 34 Abcam ab6152 1:500 o/n 4°C. 5% anti-mouse milk Bim-EL 23 Millipore AB17003 1:1000 o/n 4° C. 5%anti-rabbit BSA Bim-EL 23 Cell 2933 1:1000 o/n 4° C. 5% anti-rabbitSignaling BSA BCL-xL 30 Cell 2762 1:2000 o/n 4° C. 5% anti-rabbitSignaling BSA PARP 116/89  Cell 9542 1:1000 o/n 4° C. 5% anti-rabbitSignaling milk Cleaved 17,19 Cell 9664X 1:1000 o/n 4° C. 5% anti-rabbitCaspase 3 Signaling milk DUSP6 48 Cell 3058S 1:1000 o/n 4° C. 5%anti-rabbit Signaling BSA pRSK1/2 90 Cell 9335 1:1000 o/n 4° C. 5%anti-rabbit pS380 Signaling BSA pRSK1/2 90 Cell 11989 1:2000 o/n 4° C.5% anti-rabbit pS380 Signaling BSA pRSK- 90 Millipore 04-419 1:40000 o/n4° C. 5% anti-rabbit T359/5363 BSA Total RSK 90 Cell 9333 1:1000 o/n 4°C. 5% anti-rabbit Signaling BSA pErk 1/2 42/44 Cell 9106S 1:500 o/n 4°C. 5% anti-mouse Signaling milk Total ERK 42/44 Cell 9102 1:2000 o/n 4°C. 5% anti-rabbit Signaling milk B-Actin 42 Sigma A5441 1:500,000 o/n 4°C. 5% anti-mouse milk

FIG. 16A-FIG. 16D, FIG. 17A-FIG. 17D, and FIG. 18A-FIG. 18D show Westernblot analyses of cells treated with BVD-523 at various concentrationsfor the following: 1) MAPK signaling components in A375 cells after 4hours; 2) cell cycle and apoptosis signaling in A375 24 hours treatmentwith various amounts of BVD-523; and 3) MAPK signaling in HCT-116 cellstreated for 4 hours. The results show that acute and prolonged treatmentwith BVD-523 in RAF and RAS mutant cancer cells in-vitro affects bothsubstrate phosphorylation and effector targets of ERK kinases. Theconcentrations of BVD-523 required to induce these changes is typicallyin the low micromolar range.

Changes in several specific activity markers are noteworthy. First, theabundance of slowly migrating isoforms of ERK kinase increase followingBVD-523 treatment; modest changes can be observed acutely, and increasefollowing prolonged treatment. While this could indicate an increase inenzymatically active, phosphorylated forms of ERK, it remains noteworthythat multiple proteins subject to both direct and indirect regulation byERK remain “off” following BVD-523 treatment. First, RSK1/2 proteinsexhibit reduced phosphorylation at residues that are strictly dependenton ERK for protein modification (T359/5363). Second, BVD-523 treatmentinduces complex changes in the MAPK feedback phosphatase, DUSP6: slowlymigrating protein isoforms are reduced following acute treatment, whiletotal protein levels are greatly reduced following prolonged BVD-523treatment. Both of these findings are consistent with reduced activityof ERK kinases, which control DUSP6 function through bothpost-translational and transcriptional mechanisms. Overall, despiteincreases in cellular forms of ERK that are typically thought to beactive, it appears likely that cellular ERK enzyme activity is fullyinhibited following either acute or prolonged treatment with BVD-523.

Consistent with these observations, effector genes that require MAPKpathway signaling are altered following treatment with BVD-523. The G1/Scell-cycle apparatus is regulated at both post-translational andtranscriptional levels by MAPK signaling, and cyclin-D1 protein levelsare greatly reduced following prolonged BVD-523 treatment. Similarly,gene expression and protein abundance of apoptosis effectors oftenrequire intact MAPK signaling, and total levels of Bim-EL increasefollowing prolonged BVD-523 treatment. As noted above, however, PARPprotein cleavage and increased apoptosis were not noted in the A375 cellbackground; this suggests that additional factors may influence whetherchanges in BVD-523/ERK-dependent effector signaling are translated intodefinitive events such as cell death and cell cycle arrest.

Consistent with the cellular activity of BVD-523, marker analysissuggests that ERK inhibition alters a variety of molecular signalingevents in cancer cells, making them susceptible to both decreased cellproliferation and survival.

In sum, FIG. 16A-FIG. 16D, FIG. 17A-FIG. 17D, and FIG. 18A-FIG. 18D showthat BVD-523 inhibits the MAPK signaling pathway and may be morefavorable compared to RAF or MEK inhibition in this setting.

Finally, properties of BVD-523 may make this a preferred agent for useas an ERK inhibitor, compared to other agents with a similar activity.It is known that kinase inhibitor drugs display unique and specificinteractions with their enzyme targets, and that drug efficacy isstrongly influenced by both the mode of direct inhibition, as well assusceptibility to adaptive changes that occur following treatment. Forexample, inhibitors of ABL, KIT, EGFR and ALK kinases are effective onlywhen their cognate target is found in active or inactive configurations.Likewise, certain of these inhibitors are uniquely sensitive to eithersecondary genetic mutation, or post-translational adaptive changes, ofthe protein target. Finally, RAF inhibitors show differential potency toRAF kinases present in certain protein complexes and/or subcellularlocalizations. In summary, as ERK kinases are similarly known to existin diverse, variable, and complex biochemical states, it appears likelythat BVD-523 may interact with and inhibit these targets in a fashionthat is distinct and highly preferable to other agents.

Example 7 Effects of BVD-523 and Benchmark ERK BRAF and MEK Inhibitorson Viability and MAPK Signalling

Single Agent Proliferation Assay

Cells were seeded in 96-well plates at the densities indicated in Table15 in McCoy's 5A containing 10% FBS and allowed to adhere overnightprior to addition of compound or vehicle control. Compounds wereprepared from DMSO stocks to give the desired final concentrations. Thefinal DMSO concentration was constant at 0.1%. Test compounds wereincubated with the cells for 96 h at 37° C., 5% CO₂ in a humidifiedatmosphere. CellTiter-Glo® reagent (Promega, Madison, Wis.) was addedaccording to manufacturer's instructions and luminescence detected usingthe BMG FLUOstar plate reader (BMG Labtech, Ortenberg, Germany). Theaverage media only background value was deducted and the data analysedusing a 4-parameter logistic equation in GraphPad Prism (GraphPadSoftware, La Jolla, Calif.).

Combination Proliferation Assay

Cells were seeded into triplicate 96-well plates at the densitiesindicated in Table 15 in McCoy's 5A containing 10% FBS and allowed toadhere overnight prior to addition of test compound or vehicle control.Combinations were tested using a 10×8 dose matrix. The final DMSOconcentration was constant at 0.2%.

Test compounds were incubated with the cells for 96 h at 37° C., 5% CO₂in a humidified atmosphere. Cells were stained with Hoechst stain andfluorescence detected as described above. The average media onlybackground value was deducted and the data analysed.

Combination interactions across the dose matrix were determined by theLoewe Additivity and Bliss independence models using Chalice™Combination Analysis Software (Horizon Discovery Group, Cambridge,Mass.) as outlined in the user manual (available atchalice.horizondiscovery.com/chalice-portal/documentation/analyzer/home.jsp).Synergy is determined by comparing the experimentally observed level ofinhibition at each combination point with the value expected foradditivity, which is derived from the single-agent responses along theedges of the matrix. Potential synergistic interactions were identifiedby displaying the calculated excess inhibition over that predicted asbeing additive across the dose matrix as a heat map, and by reporting aquantitative ‘Synergy Score’ based on the Loewe model. The single agentdata derived from the combination assay plates were presented asdose-response curves generated in Chalice™.

TABLE 15 Cell Line Seeding Density Seeding density (cells/well) 96-well10 cm dish Cell Line Proliferation 6-Well Western Westerns RKO Parental1000   1 × 10⁶ 2.9 × 10⁶ RKO MEK1 1250 Not tested Not tested (Q56P/+)Clone 1 RKO MEK1 1000 7.5 × 10⁵   2 × 10⁶ (Q56P/+) Clone 2Western Blotting

Cells were seeded into 6-well plates (Experiment 1) or 10 cm dishes(Experiment 2) at the densities indicated in Table 15 in McCoy's 5Acontaining 10% FBS and allowed to adhere overnight prior to addition ofcompound or vehicle control. Test compounds were added and incubatedwith the cells for 4 or 24 h at 37° C., 5% CO₂ in a humidifiedatmosphere. Cells were harvested by trypsinisation, pelleted bycentrifugation and snap frozen on dry ice.

Lysates were prepared using RIPA buffer (50 mM Tris-hydrochloride, pH8.0; 150 mM sodium chloride; 1.0% Igepal CA-630 (NP-40); 0.5% sodiumdeoxycholate; 0.1% sodium dodecyl sulphate; 1× complete EDTA-freeprotease inhibitor cocktail (Roche, Nutley, N.J.; cat 05 892 791 001);1× phosSTOP phosphatase inhibitor cocktail (Roche Nutley, N.J.; cat. 04906 837 001)) and clarified by centrifugation at 11,000 rpm for 10 minin a bench-top centrifuge.

Total protein in the lysates was quantitated by BCA assay according tothe manufacturer's instructions (Pierce™ BCA Protein Assay Kit; ThermoScientific, Waltham, Mass.; cat. 23225), boiled in sample buffer (NuPAGELDS Sample Buffer; (Invitrogen, Carlsbad, Calif.; cat. NP0007)) andstored at −80° C.

Equal amounts of protein (40 μg) were resolved on NuPAGE 4-12% Bis-Trisgels (Invitrogen, Carlsbad, Calif.; cat. WG1402BOX) and blotted ontoPVDF membranes using iBlot gel transfer stacks (Invitrogen, Carlsbad,Calif.; cat. IB4010-01) on an iBlot gel transfer device (InvitrogenCarlsbad, Calif.) according to the manufacturer's instructions.

Blots were probed using the antibodies and block conditions detailed inTable 16. Western blots were developed using Pierce™ ECL2 Westernblotting substrate (Thermo Scientific, Waltham, Mass.; cat. 80196) andimaged using a FluorChem M Western blot imager (ProteinSimple, San Jose,Calif.).

TABLE 16 Antibodies and Western Blotting Conditions SizeIncubation/block Antigen (kDa) Supplier Cat No Dilution ConditionsSecondary pRSK-T359/S363 90 Millipore 04-419 1:20000 o/n 4° C. 5% BSAanti-rabbit Total RSK 90 Cell Signaling 9333 1:1000 o/n 4° C. 5% BSAanti-rabbit pErk 1/2 42/44 Cell Signaling 9106S 1:500 o/n 4° C. 5% milkanti-mouse Total ERK 42/44 Cell Signaling 9102 1:2000 o/n 4° C. 5% milkanti-rabbit pMEK 1/2 45 Cell Signaling 9154 1:1000 o/n 4° C. 5% BSAanti-rabbit Total MEK 45 Cell Signaling 9126 1:1000 o/n 4° C. 5% BSAanti-rabbit DUSP6 48 Cell Signaling 3058S 1:1000 o/n 4° C. 5% BSAanti-rabbit pRB (Ser780) 105 Cell Signaling 9307 1:2000 o/n 4° C. 5% BSAanti-rabbit CCND1 34 Abeam ab6152 1:500 o/n 4° C. 5% milk anti-mouseB-Actin 42 Sigma A5441 1:100,000 o/n 4° C. 5% milk anti-mouseAnti-rabbit — Cell Signaling 7074S 1:2000 1 h room temp; —HRP-conjugated Block matched to secondary primary Antibody Anti-mouse —Cell Signaling 7076 1:5000 1 h room temp; — HRP-conjugated Block matchedto secondary primary Antibody

The MEK1 (Q56P) mutation exemplifies a class of clinically relevantMEK1/2 activating mutations known to up-regulate the MAPK pathway anddrive acquired resistance to BRAF or MEK inhibitors.

This study used a pair of RKO BRAF(V600E) cell lines that are isogenicfor the presence or absence of a MEK1 (Q56P) activating mutation, toassess the effect that activating MEK mutations have in response to thenovel ERK inhibitor BVD-523 versus other benchmark MAPK inhibitors.

Effects of on cell viability were assessed by quantitating cellular ATPlevels using CellTiter-Glo® after 96 h. Single agent assays demonstratedthat the double mutant BRAF(V600E)::MEK1(Q56P) cells displayed amarkedly reduced sensitivity to inhibition with benchmark clinical BRAF(exemplified by Dabrafenib) or MEK (exemplified by Trametinib)inhibitors relative to the parental BRAF(V600E) cells, whichdemonstrates the suitability of this isogenic model for recapitulatingthe acquired resistance known to be associated with this class ofmutation in the clinic (Table 17).

TABLE 17 Single Agent IC₅₀ Values RKO RKO MEK1 Q56P/+ RKO MEK1 Q56P/+Compound Parental Cl.1 Cl.2 BVD-523 0.20 0.17 0.18 SCH772984 0.04 0.140.12 Dabrafenib n.d. n.d. n.d. Trametinib 0.006 0.093 0.080 Paclitaxel0.002 0.002 0.002 n.d.—not determined, only a partial dose responseachieved

In contrast, response to BVD-523 was identical in both the parental anddouble mutant cells, indicating that BVD-523 is not susceptible to thismechanism of acquired resistance.

These results were identical in two independently derived double mutantBRAF(V600E)::MEK1(Q56P) cell line clones confirming that thesedifferences in response versus the parental cells were specificallyrelated to the presence of the MEK1 mutation rather than an unrelatedclonal artifact (FIG. 22A-FIG. 22E). Similar results were also observedwith a second mechanistically distinct benchmark ERK inhibitor(SCH772984), which supports the notion that these observations arespecifically related to inhibition of ERK and not due to an off-targeteffect.

The effect of combining BVD-523 with a BRAF inhibitor (exemplified byDabrafenib) was also assessed in these cell lines across a matrix ofconcentrations using the Loewe Addivity or Bliss Independence modelswith Horizon's Chalice™ combination analysis software (FIG. 23-FIG. 23Oand FIG. 24A-FIG. 24O). The presence of potentially synergisticinteractions was then assessed by displaying the calculated excessinhibition over that predicted as being additive across the dose matrixas a heat map, and by calculating a ‘Volume Score’ that shows whetherthe overall response to a combination is synergistic (positive values),antagonistic (negative values) or additive (˜0).

The results suggest that the BVD-523::Dabrafenib combination was mainlyadditive in the parental and mutant cell line. In contrast, thecombination of a MEK inhibitor (trametinib) plus Dabrafenib, while beingmostly additive in the parental cell line, showed strong synergy in thedouble mutant BRAF(V600E)::MEK1(Q56P) cell line (FIG. 25A-FIG. 25O).Loewe Volumes, Bliss Volumes and Synergy scores for the combinationstested are shown in Tables 18-20, respectively and are shown graphed inFIG. 26A-FIG. 26C.

TABLE 18 Loewe Volumes RKO MEK1 RKO MEK1 (Q56P) - (Q56P) - RKO ParentalClone 1 Clone 2 BVD-523 × Dabrafenib 3.54 2.88 2.35 Dabrafenib ×SCH772984 5.7 6.79 6.14 Dabrafenib × Trametinib 5.68 12.6 11.6

TABLE 19 Bliss Volumes RKO MEK1 RKO MEK1 (Q56P) - (Q56P) - RKO ParentalClone 1 Clone 2 BVD-523 × Dabrafenib −0.894 0.527 1.42 Dabrafenib ×SCH772984 0.209 4.3 5.07 Dabrafenib × Trametinib 0.353 10.8 9.87

TABLE 20 Synergy Scores RKO MEK1 RKO MEK1 (Q56P) - (Q56P) - RKO ParentalClone 1 Clone 2 BVD-523 × Dabrafenib 3.18 2.31 1.77 Dabrafenib ×SCH772984 4.56 5.57 4.36 Dabrafenib × Trametinib 5.58 11 9.83

Effects on MAPK pathway signally was assessed by Western blotting. Thelevels of basal ERK phosphorylation (DMSO samples) was markedlyup-regulated in the MEK1(Q56P)-expressing line relative to parentalfurther confirming that this isogenic model faithfully recapitulates theexpected phenotype for the expression of MEK activating acquiredresistance mutations.

In the parental BRAF(V600E) RKO cells, a reduced level of RSK1/2phosphorylation is observed following acute treatment with RAF, MEK andERK kinase inhibitors at pharmacologically active concentrations. Incontrast, isogenic, double mutant BRAFV600E::MEK1Q56P cells do notexhibit reduced RSK phosphorylation following BRAF or MEK inhibitortreatment, while BVD-523 remains effective at similar concentrations(FIG. 27A-FIG. 27I). The dotted lines indicate that thetrametinib-treated samples (plus matched DMSO control) and blots arederived from a separate experiment to the BRAFi and BVD-523 treatedsamples.

Changes in effector gene signaling consistent with cell growthinhibition patterns are observed following prolonged inhibitortreatment. In parental RKO lines, a reduced level of phosphorylated pRBis observed following prolonged MEK and ERK inhibitor treatment. At thelevel of pRB modulation, MEK1 mutant lines appear insensitive to lowconcentration MEK inhibitor treatment, while higher concentrationsremain effective. Critically, BVD-523 potency against pRB activity doesnot appear to be strongly affected by MEK mutation. Surprisingly, RAFinhibitor treatment does not affect pRB status, despite potentinhibition of upstream signaling, in both parental and MEK mutantbackgrounds.

In summary, these results show that BVD-523 is not susceptible toacquired resistance driven by MEK activating mutations such as MEK1(Q56P). In addition they suggest that in combination the interactionsbetween BVD-523 and BRAFi (exemplified by Dabrafenib) are additiveirrespective of the presence of a MEK activating mutation.

Example 8 Combination Interactions Between ERK Inhibitors

RAF mutant melanoma cell line A375 cells were cultured in DMEM with 10%FBS and seeded into triplicate 96-well plates at an initial density of2000 cells per well. Combination interactions between ERK inhibitorsBVD-523 and SCH772984 were analized after 72 hours as described above inExample 4. Viability was determined using CellTiter-Glo® reagent(Promega, Madison, Wis.) according to manufacturer's instructions andluminescence was detected using the BMG FLUOstar plate reader (BMGLabtech, Ortenberg, Germany).

Visualization of the Loewe and Bliss ‘excess inhibition’ heat mapssuggested that the combination of BVD-523 and SCH772984 was mainlyadditive with windows of potential synergy in mid-range doses (FIG.28A-FIG. 28E).

In summary, these results suggest that interactions between BVD-523 andSCH772984 are at least additive, and in some cases synergistic.

Example 9 Targeting the MAPK Signaling Pathway in Cancer: PromisingActivity with the Novel Selective ERK1/2 Inhibitor BVD-523 (Ulixertinib)

Treatment strategies for cancer have evolved from classiccytotoxic-based approaches to agents that counteract the effects ofgenetic lesions that drive aberrant signaling essential to tumorproliferation and survival. For example, the ERK module of themitogen-activated protein kinase (MAPK) signaling cascade(RAS-RAF-MEK-ERK) (Cargnello and Rouxx 2011) can be engaged by severalreceptor tyrosine kinases (e.g., EGFR and ErbB-2) in addition toconstitutively activated mutations of pathway components such as RAS andBRAF (Gollob et al. 2006). Through aberrant activation of ERK signaling,genetic alterations in RAS or BRAF result in rapid tumor growth,increased cell survival, and resistance to apoptosis (Poulikakos et al.2011, Corcoran et al. 2010, Nazarian et al. 2010, Shi et al. 2014, Wagleet al. 2011). Activating mutations of RAS family members KRAS and NRASare found in ˜30% of all human cancers, with particularly high incidencein pancreatic (Kanda et al. 2012) and colorectal cancer (Arrington etal. 2014). Constitutively activating mutations in the BRAF gene thatnormally encodes for valine at amino acid 600 have been observed inmelanoma, thyroid carcinoma, colorectal cancer, and non-small cell lungcancer (Hall et al. 2014). Cancers bearing genetic mutations that resultin changes of the downstream components ERK and MEK have also beenreported (Ojesina et al. 2014, Arcila et al. 2015). Alterations thatactivate the MAPK pathway are also common in the setting of resistanceto targeted therapies (Groenendijk et al. 2014). Thus, targeting theMAPK pathway terminal master kinases (ERK1/2) is a promising strategyfor tumors harboring such pathway activating alterations (e.g., BRAF,NRAS, and KRAS).

Three MAPK pathway-targeting drugs have been approved by the US Food andDrug Administration (FDA) for single-agent treatment of nonresectable ormetastatic cutaneous melanoma with BRAF^(V600) mutations: the BRAFinhibitors vemurafenib and dabrafenib and the MEK inhibitor trametinib.Furthermore, the combination of dabrafenib and trametinib is alsoapproved in this indication (Queirolo et al. 2015 and Massey et al.2015). An additional MEK inhibitor, cobimetinib, is approved in thisindication as part of a combination regimen with BRAF inhibitors.Clinical experience with these drugs validates the MAPK pathway as atherapeutic target. In phase III trials of patients withBRAF^(V600)-mutant melanoma, the single agents vemurafenib anddabrafenib demonstrated superior response rates (approximately 50% vs.5-19%) and median progression-free survival (PFS, 5.1-5.3 months vs.1.6-2.7 months) over cytotoxic chemotherapy (dacarbazine) (Chapman etal. 2011 and Hauschild et al. 2012). Furthermore, clinical use ofconcomitant BRAF-plus MEK-targeted therapies has demonstrated thatsimultaneous targeting of different nodes in the MAPK pathway canenhance the magnitude and duration of response. First-line use of BRAFplus MEK-targeted agents (dabrafenib/trametinib orcobimetinib/vemurafenib) further improved median overall survivalcompared with single-agent BRAF inhibition (Robert et al. 2015, Long etal. 2015, Larkin et al. 2014). Thus, combined BRAF-/MEK-targeted therapyis a valuable treatment option for patients with metastatic melanomawith BRAF^(V600) mutations.

Despite improvements in clinical outcomes seen with BRAF-/MEK-inhibitorcombination therapies, durable benefit is limited by the eventualdevelopment of acquired resistance and subsequent disease progression,with median PFS ranging from approximately 9 to 11 months. (Robert etal. 2015, Long et al. 2015, Larkin et al. 2014, and Flaherty et al.2012). Genetic mechanisms of acquired resistance to single-agent BRAFinhibition have been intensely studied, and identification of resistancemechanisms include splice variants of BRAF (Poulikakos et al. 2011),BRAF^(V600E) amplification (Corcoran et al. 2010), MEK mutations (Wagleet al. 2014), NRAS mutations, and RTK activation (Nazarian et al. 2010and Shi et al. 2014). Resistance mechanisms in the setting ofBRAF-/MEK-inhibitor combination therapy are beginning to emerge andmirror that of BRAF single-agent resistance (Wagle et al. 2014 and Longet al. 2014). These genetic events all share in common the ability toreactivate ERK signaling. Indeed, reactivated MAPK pathway signaling asmeasured by ERK transcriptional targets is common in tumor biopsies fromBRAF inhibitor-resistant patients (Rizos et al. 2014). Furthermore,ERK1/2 reactivation has been observed in the absence of a geneticmechanism of resistance (Carlino et al. 2015). Therefore, the quest toachieve durable clinical benefit has led researchers to focus onevaluating additional agents that target the downstream MAPK componentsERK1/2. Inhibiting ERK may provide important clinical benefit topatients with acquired resistance to BRAF/MEK inhibition. ERK familykinases have shown promise as therapeutic targets in preclinical cancermodels, including those cancers resistant to BRAF or MEK inhibitors(Morris et al. 2013 and Hatzivassiliou et al. 2012). However, thepotential use of such ERK1/2 inhibitors expands beyondacquired-resistance in melanoma.

Targeting ERK1/2 is a rational strategy in any tumor type harboringknown drivers of MAPK, not only BRAF/MEK therapy-relapsed patients. AsERK1 and ERK2 reside downstream in the pathway, they represent aparticularly attractive treatment strategy within the MAPK cascade thatmay avoid upstream resistance mechanisms. Here, preclinicalcharacterization of BVD-523 (ulixertinib) in models of MAPKpathway-dependent cancers is reported, including drug-naïve and BRAF/MEKtherapy acquired-resistant models. Results of a phase I dose-findingstudy of BVD-523 are included as a companion publication in thisjournal. See, Examples 17-24.

In the present invention, BVD-523 was shown to be a potent, highlyselective, reversible, small molecule ATP-competitive inhibitor ofERK1/2 with in vitro and in vivo anticancer activity.

BVD-523 (ulixertinib) was identified and characterized as a novel,reversible, ATP-competitive ERK1/2 inhibitor with high potency andERK1/2 selectivity. BVD-523 caused reduced proliferation and enhancedcaspase activity, most notably in cells harboring MAPK (RAS-RAF-MEK)pathway mutations. In in vivo BRAF^(V600E) xenograft studies, BVD-523showed dose-dependent growth inhibition and tumor regressions.Interestingly, BVD-523 inhibited phosphorylation of target substratesdespite increased phosphorylation of ERK1/2. BVD-523 also demonstratedantitumor activity in models of acquired resistance to single-agent andcombination BRAF/MEK targeted therapy. Synergistic antiproliferativeeffects in a BRAF^(V600E)-mutant melanoma cell line xenograph model werealso demonstrated when BVD-523 was used in combination with BRAFinhibition. These studies suggest that BVD-523 holds promise as atreatment for ERK-dependent cancers, including those whose tumors haveacquired resistance to other treatments targeting upstream nodes of theMAPK pathway.

Example 10 Discovery and Initial Characterization of a Novel ERK1/2Inhibitor, BVD-523 (Ulixertinib)

Following extensive optimization of leads originally identified using ahigh-throughput, small-molecule screen (Aronov et al. 2009), a noveladenosine triphosphate (ATP)-competitive ERK1/2 inhibitor, BVD-523(ulixertinib) was identified (FIG. 29 A). BVD-523 is a potent ERKinhibitor with a K_(i) of 0.04±0.02 nM against ERK2. It was shown to bea reversible, competitive inhibitor of ATP, as the IC₅₀ values for ERK2inhibition increased linearly with increasing ATP concentration (FIG.29B and FIG. 29C). The IC₅₀ remained nearly constant for incubationtimes minutes, suggesting rapid equilibrium and binding of BVD-523 withERK2 (FIG. 29D). BVD-523 is also a tight-binding inhibitor ofrecombinant ERK1 (Rudolph et al. 2015), exhibiting a K_(i) of <0.3 nM.

Binding of BVD-523 to ERK2 was demonstrated using calorimetric studiesand compared to data generated using the ERK inhibitors SCH772984 andpyrazolylpyrrole (Arovov et al. 2007). All compounds bound andstabilized inactive ERK2 with increasing concentration, as indicated bypositive ΔTm values (FIG. 29E). The 10- to 15-degree change in ΔTmobserved with BVD-523 and SCH-772984 is consistent with compounds thathave low-nanomolar binding affinities (Fedorov et al. 2012). BVD-523demonstrated a strong binding affinity to both phosphorylated activeERK2 (pERK2) and inactive ERK2 (FIG. 29F). A stronger affinity to pERK2compared with inactive ERK2 was observed. BVD-523 did not interact withthe negative control protein p38a MAP kinase (FIG. 29F).

BVD-523 demonstrated excellent ERK1/2 kinase selectivity based onbiochemical counter-screens against 75 kinases in addition to ERK1 andERK2. The ATP concentrations were approximately equal to the K_(m) inall assays. Kinases inhibited to greater than 50% by 2 μM BVD-523 wereretested to generate K_(i) values (or apparent Ki; Table 21). Twelve ofthe 14 kinases had a K_(i) of <1 μM. The selectivity of BVD-523 for ERK2was >7000-fold for all kinases tested except ERK1, which was inhibitedwith a Ki of <0.3 nM (10-fold). Therefore, BVD-523 is a highly potentand selective inhibitor of ERK1/2.

TABLE 21 BVD-523 displays selectivity for ERK1 and ERK2 kinases. KinaseKi (μM) CDK1/cyclinB 0.07^(a) CDK2/cyclinA 0.36 CDK5/p35 0.09^(a)CDK6/cycinD3 0.09^(a) ERK1 0.0003 ERK2 0.00004 GSK3b 0.32 JNK2α 0.65^(a)JNK3 1.3 P38γ 0.45^(a) P38δ 0.24^(a) ROCKI 11.1 ROCKII 0.27^(a) RSK30.45 ^(a)Apparent. <50% inhibition at 2 μM: ABL, AKT3, AMPK, AUR1, AUR2,AXL, BLK, CAMKII, CAMKIV, CHK1, CHK2, CK1, CK2, CSK, EGFR, EPHB4, FES,FGFR3, FLT3, FYN, IGF1R, IKKα, IKKβ, IKKi, IRAK4, IRTK, ITK, JAK3,JNK1α1, KDR, LCK, LYN, cMET, MKK4, MKK6, MKK7β, MLK2, MSK1, MST2, NAK,NEK2, p38α, p38β, p70S6K, PAK2, PDGFRα, PDK1, PKA, PKCα, PKCβII, PKCγ,PKCi, PKCθ, PRAK, PRK2, cRAF, SGK, SRC, SYK, TAK1, TIE2, ZAP70

Example 11 BVD-523 Preferentially Inhibits Cellular Proliferation andEnhances Caspase-3/7 Activity In Vitro in Cancer Cell Lines with MAPKPathway-Activating Mutations

BVD-523 cellular activity was assessed in a panel of approximately 1,000cancer cell lines of various lineages and genetic backgrounds (FIG. 30Aand Table 22). Cell lines were classified as MAPK wild type (wt) ormutant depending on the absence or presence of mutations in RAS familymembers and BRAF. Although some MAPK-wt cell lines were sensitive toBVD-523, generally BVD-523 inhibited proliferation preferentially incells with MAPK pathway alterations.

Next, the growth and survival impact of BVD-523 treatment on sensitivecells was characterized. Fluorescence activated cell sorting (FACS)analysis was performed on BRAF^(V600E)-mutant melanoma cell line UACC-62following treatment with BVD-523 at 500 nM or 2000 nM for 24 hours.Treated cells were arrested in the G1 phase of the cell cycle in aconcentration-dependent manner (FIG. 30B).

In addition, caspase-3/7 activity was analyzed as a measure of apoptosisin multiple human cancer cell lines. A concentration- andcell-line-dependent increase in caspase 3/7 was observed followingtreatment with BVD-523 for 72 hours (FIG. 30C). BVD-523 treatmentresulted in pronounced caspase-3/7 induction in a subset ofMAPK-activated cell lines harboring a BRAF^(V600) mutation (A375, WM266,and LS411N). This is consistent with earlier observations forpreferential inhibition of proliferation by BVD-523 in MAPKpathway-mutant cancer cell lines (FIG. 30A).

To further characterize the mechanism of action and effects on signalingelicited by BVD-523, the levels of various effector and MAPK-relatedproteins were assessed in BVD-523-treated BRAF^(V600E)-mutant A375melanoma cells (FIG. 30D). Phospho-ERK1/2 levels increased in aconcentration-dependent manner after 4 and 24 hours of BVD-523treatment. Despite prominent concentration-dependent increases inpERK1/2 observed with 2 μM BVD-523 treatment, phosphorylation of theERK1/2 target RSK1/2 was reduced at both 4 and 24 hours, which isconsistent with sustained inhibition. Total protein levels of DUSP6, adistal marker of ERK1/2 activity, were also attenuated at 4 and 24hours. Following 24 hours of treatment with BVD-523, the apoptoticmarker BIM-EL increased in a dose-dependent manner, while cyclin D-1 andpRB was attenuated at 2 μM. All effects are consistent with on-targetERK1/2 inhibition.

TABLE 22 Viability ratio Cell Compound Fitted 20 10 5 2.5 1.25 0.6250.3125 0.15625 0.078125 Barcode Organ ID Cell Line No MGH_IC50 uM uM uMuM uM uM uM uM uM 026_8049_00277140 Biliary Tract 8049 ETK-1 4563.525905 0.587 0.805 0.874 0.9304 0.8796 0.954 1.0285 1.094 0.9918026_664_00277150 Biliary Tract 664 HuCCT1 456 3.600435 0.663 0.734 0.8470.9661 0.9652 1.029 0.9656 1.0172 0.9981 026_653_00278500 Biliary Tract653 EGI-1 456 4.229085 0.693 0.686 0.74 0.7843 0.8546 0.889 0.967 0.92860.9525 026_8204_00278540 Biliary Tract 8204 TGBC24TKB 456 5.609877 0.7680.875 0.826 0.8122 0.8629 0.862 0.8909 0.9353 0.9353 026_8188_00293390Biliary Tract 8188 TGBC1TKB 456 6.179372 0.915 0.929 0.913 0.9808 0.92011.144 1.0128 0.9048 0.9496 026_330_00278580 Bone 330 H-EMC-SS 4560.038629 0.36 0.392 0.383 0.4615 0.437 0.566 0.7772 0.9442 0.9662026_8047_00283120 Bone 8047 ES7 456 1.846677 0.515 0.521 0.539 0.53620.7569 0.769 0.8353 0.9371 0.9503 026_8053_00287650 Bone 8053 EW-13 4562.197657 0.333 0.545 0.665 0.8165 0.9099 0.962 0.9571 0.9945 1.0406026_8227_00288230 Bone 8227 CADO-ES1 456 2.29467 0.359 0.554 0.5851.0389 0.9034 0.952 1.1264 1.2671 1.0023 026_8050_00279380 Bone 8050EW-1 456 2.409222 0.487 0.555 0.593 0.6879 0.6908 0.788 0.8057 0.8920.9331 026_306_00278530 Bone 306 SK-ES-1 456 2.4607 0.527 0.556 0.5770.682 0.673 0.855 0.8706 0.8641 0.8867 026_305_00277180 Bone 305 U-2 OS456 2.847932 0.145 0.599 0.657 0.7441 0.8231 0.793 0.8449 0.9969 0.8925026_337_00283440 Bone 337 HuO9 456 2.916396 0.448 0.696 0.939 0.80260.8478 0.903 0.9796 0.8682 1.0954 26_8227_00304340 Bone 8227 CADO-ES1456 2.975471 0.542 0.666 0.884 0.8971 0.9918 0.974 1.0247 1.0031 1.0728026_8043_00283110 Bone 8043 ES1 456 2.981717 0.543 0.663 0.705 0.78650.811 0.835 0.7995 0.9278 0.8292 026_8142_00282550 Bone 8142 NOS-1 4563.203574 0.547 0.775 0.825 0.892 0.7556 0.894 0.8477 1.1038 0.9881026_8055_00290580 Bone 8055 EW-18 456 3.311765 0.638 0.688 0.718 1.10120.9293 0.993 1.0646 1.1078 1.0895 026_8058_00293350 Bone 8058 EW-3 4563.365484 0.715 0.595 0.72 0.7195 0.8774 0.8 0.8628 0.9101 1.2148026_339_00277160 Bone 339 NY 456 3.400937 0.59 0.789 0.875 0.9222 0.95940.975 0.933 1.1433 0.9637 026_8165_00287690 Bone 8165 SK-PN-DW 4563.476926 0.621 0.812 0.933 0.9706 0.9188 0.991 1.0026 1.0012 1.0432026_326_00282540 Bone 326 MHH-ES-1 456 3.524605 0.618 0.719 0.824 0.82350.8745 0.966 1.1187 1.0416 0.913 026_8048_00279370 Bone 8048 ES8 4563.530755 0.572 0.698 0.765 0.8053 0.8255 0.912 0.931 0.9069 0.9095026_331_00278590 Bone 331 HOS 456 3.602447 0.53 0.798 0.749 0.76090.8727 0.839 0.8786 0.8236 0.972 026_8045_00282660 Bone 8045 ESS 4563.665641 0.704 0.653 0.874 0.8827 0.7407 0.913 0.8294 1.0585 1.162026_8059_00283090 Bone 8059 EW-7 456 3.827217 0.662 0.72 0.76 0.8310.8857 0.95 0.9309 0.9266 0.9963 026_8201_00282520 Bone 8201 ES3 4563.982944 0.707 0.721 0.778 0.8414 0.8385 0.989 0.9588 1.0248 1.0261026_8056_00314310 Bone 8056 EW-22 456 4.196343 0.701 0.827 0.819 0.88240.9413 0.894 0.9848 1.0221 1.1139 026_329_00282700 Bone 329 G-292 Clone456 4.264519 0.705 0.918 0.893 0.949 1.0435 1.016 0.9814 0.9215 1.1209A141B1 026_324_00278550 Bone 324 CAL-72 456 4.286956 0.731 0.942 0.9420.9262 0.9863 0.966 0.9797 0.9547 1.0113 026_304_00283460 Bone 304Saos-2 456 4.597501 0.742 0.784 0.9 0.8765 0.9306 0.926 0.9516 0.951.0352 026_325_00283060 Bone 325 CAL-78 456 4.650689 0.727 0.913 0.9180.9149 0.8999 0.897 0.9065 1.0242 1.0181 026_1138_00278560 Bone 1138 CS1456 4.765346 0.744 0.848 0.864 0.8601 0.8818 1.016 1.0005 0.9749 0.977026_8162_00282560 Bone 8162 SJSA-1 456 4.867747 0.802 0.803 0.805 0.77650.762 0.974 1.0651 1.0412 1.0623 026_336_00283430 Bone 336 HuO-3N1 4564.900372 0.724 0.86 0.806 0.8427 0.8308 0.87 0.8972 0.9772 1.0073026_328_00278600 Bone 328 TC-71 456 5.117725 0.844 0.939 0.97 1.02210.9857 1.072 1.023 1.0842 1.0771 026_8054_00282530 Bone 8054 EW-16 4565.180986 0.835 0.775 0.925 0.8021 0.8155 0.934 0.9339 1.058 1.1414026_335_00308220 Bone 335 MG-63 456 5.257203 0.884 0.841 0.852 0.87451.0185 1.081 1.1007 1.0325 1.0709 026_1241_00283070 Bone 1241 CHSA8926456 5.394244 0.883 0.851 0.947 0.9788 0.9327 1.019 1.0879 1.0294 1.119026_8044_00279340 Bone 8044 ES4 456 6.158016 0.822 0.876 0.92 0.86120.8592 0.873 0.8969 0.9914 0.9632 026_8057_00283080 Bone 8057 EW-24 4566.273162 0.927 0.899 0.97 0.9817 0.9704 1.012 0.9822 0.9596 0.9793026_8051_00285230 Bone 8051 EW-11 456 6.340509 0.924 1.008 0.846 0.93091.047 0.868 0.9762 1.0834 1.048 026_8046_00279351 Bone 8046 ES6 4566.745328 0.882 1.065 1.046 1.0203 1.0617 0.85 0.8881 1.0547 0.9578026_8146_00285140 Brain 8146 ONS-76 456 1.017095 0.362 0.369 0.387 0.520.6146 0.733 0.8773 0.9176 1.1016 026_8009_00285111 Brain 8009 AM-38 4562.859683 0.545 0.617 0.664 0.5881 0.5592 0.692 0.826 0.9522 1.078026_8091_00285281 Brain 8091 KS-1 456 2.979142 0.617 0.59 0.62 0.70570.7844 0.875 0.9025 1.0584 1.1343 026_388_00285240 Brain 388 MOG-G-CCM456 3.029922 0.505 0.754 0.852 0.977 0.9597 0.989 1.1211 1.0361 1.0747026_352_00283150 Brain 352 LN-229 456 3.053877 0.552 0.647 0.662 0.72060.8119 0.932 0.9285 1.0381 1.0748 026_8214_00290680 Brain 8214 YH-13 4563.068585 0.558 0.628 0.773 1.0443 0.9614 1.113 1.1139 1.093 1.2619026_8214_00288290 Brain 8214 YH-13 456 3.531592 0.627 0.651 0.866 0.83870.8073 0.922 1.077 1.0619 1.1182 026_358_00293700 Brain 358 D283 Med 4563.551231 0.64 0.743 0.916 0.9078 0.9464 0.928 0.871 0.9767 1.4204026_8061_00290830 Brain 8061 GB-1 456 3.686496 0.613 0.692 0.809 0.82550.8977 0.877 0.9922 0.9416 0.8923 026_374_00283180 Brain 374 U-251 MG456 3.933399 0.654 0.733 0.861 0.8164 0.8981 0.891 0.9222 0.9976 1.1342026_343_00283160 Brain 343 PFSK-1 456 3.963833 0.626 0.904 1.007 1.02290.934 1.078 1.0411 1.0084 1.052 026_393_00283190 Brain 393 YKG-1 4563.987729 0.646 0.714 0.77 0.8178 0.8374 0.848 0.953 0.8805 1.0209026_8028_00287630 Brain 8028 D-263MG 456 4.157483 0.662 0.709 0.7340.7813 0.8341 0.839 0.9023 0.9333 1.0435 026_379_00283140 Brain 379 GAMG456 4.214093 0.681 0.701 0.829 0.8043 0.8412 0.83 0.9322 0.8908 1.046026_8019_00293320 Brain 8019 CAS-1 456 4.309856 0.732 0.772 0.825 0.92220.9783 1.227 0.8865 0.8407 1.1628 026_8001_00285100 Brain 8001 8-MG-BA456 4.345495 0.688 0.845 0.88 0.8958 0.9285 0.912 0.9148 0.9398 1.07026_351_00283450 Brain 351 LN-18 456 4.481815 0.685 0.822 0.862 0.87460.8966 0.902 0.9022 0.9311 0.9325 026_357_00283410 Brain 357 H4 4564.481909 0.721 0.808 0.876 0.858 0.8822 0.94 1.0232 0.9287 1.0614026_8085_00293731 Brain 8085 KINGS-1 456 4.48888 0.797 0.781 0.82 0.96450.9641 1.015 0.9981 1.0442 0.974 026_350_00284910 Brain 350 M059J 4564.537622 0.723 0.834 0.869 0.8888 0.9473 0.909 0.9144 1.0191 1.0818026_8015_00308070 Brain 8015 Becker 456 4.548091 0.696 0.814 0.8760.8121 0.825 0.889 0.917 0.9113 1.0138 026_8160_00287680 Brain 8160SF539 456 4.738405 0.726 0.919 0.827 0.8875 0.9392 0.934 0.952 1.05250.9285 026_8159_00287670 Brain 8159 SF268 456 4.804304 0.786 0.825 0.9230.9225 0.9772 0.99 0.978 0.9754 0.9699 026_359_00283100 Brain 359 Daoy456 4.81575 0.683 0.74 0.796 0.8102 0.876 0.896 0.9135 0.9254 0.9257026_8217_00290890 Brain 8217 SK-MG-1 456 4.83361 0.725 0.784 0.83 0.8440.8849 0.901 0.9119 0.9353 0.9859 026_342_00285160 Brain 342 SW 1783 4564.84471 0.796 0.905 0.896 0.9349 1.0662 0.935 1.0347 1.0148 1.08026_8029_00288240 Brain 8029 D-336MG 456 4.925588 0.792 0.892 0.9970.8872 0.9556 0.944 1.105 1.0724 1.1057 026_8030_00295500 Brain 8030D-392MG 456 4.966575 0.791 0.866 0.824 0.9331 0.8638 1.022 0.9531 0.96881.0531 026_8089_00287440 Brain 8089 KNS-81-FD 456 5.077406 0.804 0.890.869 0.8138 0.8421 1.049 1.0747 0.9317 1.1222 026_8138_00285290 Brain8138 NMC-G1 456 5.086457 0.771 0.817 0.763 0.762 0.8138 0.887 0.9681.0104 1.049 026_8139_00285130 Brain 8139 no-10 456 5.147267 0.824 0.8880.928 0.9069 0.9436 1.031 0.943 1.05 1.0543 026_8083_00293720 Brain 8083KALS-1 456 5.203248 0.817 0.765 0.902 0.9843 0.9376 0.915 0.9382 1.01160.9496 026_378_00284880 Brain 378 DK-MG 456 5.2953 0.789 0.757 0.7130.7178 0.7297 0.849 0.8425 0.9101 1.0114 026_383_00284900 Brain 382LN-405 456 5.313289 0.839 0.886 0.901 0.937 0.8868 1.041 0.949 1.08161.0986 026_8032_00293340 Brain 8032 D-542MG 456 5.342096 0.822 0.8560.927 0.9108 0.8977 0.934 0.9191 0.9743 0.9895 026_344_00282720 Brain344 LNZTA3WT4 456 5.43481 0.777 1.136 0.931 0.9386 0.9371 1.018 1.04411.0001 0.9944 026_8167_00290910 Brain 8167 SNB75 456 5.474524 0.76 0.7720.83 0.8023 0.8147 1.01 0.8804 1.0523 0.9382 026_8087_00285270 Brain8087 KNS-42 456 5.484622 0.862 0.858 0.846 0.8777 0.9301 0.972 0.99231.0701 1.0135 026_354_00287481 Brain 354 U-87 MG 456 5.588679 0.8510.949 0.942 0.9605 0.9939 1.004 1.0222 0.9964 1.0855 026_8140_00285300Brain 8140 no-11 456 5.608459 0.844 0.885 0.896 0.959 0.9411 0.8930.9627 1.0352 0.9432 026_8221_00284860 Brain 8221 D-423MG 456 5.7313720.807 0.783 0.798 0.8469 0.8833 0.945 0.9877 0.9131 0.9215026_348_00283400 Brain 348 DBTRG-05MG 456 5.749405 0.792 0.75 0.7630.7919 0.7917 0.856 0.9393 0.9013 0.9922 026_341_00285310 Brain 341 SW1088 456 5.805148 0.869 0.907 0.895 0.8893 0.9105 0.919 0.966 0.89611.0957 026_356_00283420 Brain 356 Hs 683 456 5.858982 0.841 0.993 0.8880.8789 0.8908 1.04 0.8743 0.906 1.058 026_8031_00287640 Brain 8031D-502MG 456 5.99897 0.825 0.819 0.771 0.7649 0.8658 0.834 0.9457 0.99220.9572 026_8224_00284870 Brain 8224 D-566MG 456 6.026403 0.839 0.840.858 0.8841 0.9252 0.941 0.968 0.9815 0.9816 026_389_00284920 Brain 389MOG-G-UVW 456 6.074777 0.861 0.876 0.879 0.9129 0.9004 0.902 1.05970.9108 1.0304 026_341_00283470 Brain 341 SW1088 456 6.105387 0.913 0.890.897 0.897 0.9463 0.931 0.9486 0.9506 1.1024 026_375_00284850 Brain 37542-MG-BA 456 6.106423 0.896 0.892 0.932 0.944 0.9633 0.976 0.9735 0.98711.0285 026_1122_00283170 Brain 1122 SF-295 456 6.112956 0.879 0.9090.909 0.9301 0.9215 0.946 0.9199 0.9433 1.0329 026_8158_00290650 Brain8158 SF126 456 6.158755 0.86 0.97 1 1.0676 1.0025 1.187 0.9851 0.96271.1324 026_340_00285250 Brain 340 CCF-STTG1 456 6.170298 0.851 0.9110.916 0.9011 0.9748 0.919 0.9109 0.9323 0.9837 026_380_00284890 Brain380 GMS-10 456 6.23472 0.842 0.885 0.853 0.8466 0.8914 0.914 0.88821.0019 1.0545 026_354_00290361 Brain 354 U-87 MG 456 6.315816 0.93 0.8740.977 0.9346 0.9336 1.084 0.9295 1.062 1.073 026_8063_00290841 Brain8063 GI-1 456 6.443002 0.809 0.877 0.913 0.9018 0.9184 0.86 0.92410.9072 0.9709 026_8027_00293330 Brain 8027 D-247MG 456 6.453915 0.9280.905 0.908 0.8398 0.8592 0.911 0.9463 0.9529 0.9044 026_346_00283390Brain 346 A172 456 6.640511 0.941 0.953 0.995 1.0208 0.9242 1.026 1.01411.0236 1.0356 026_8089_00291170 Brain 8089 KNS-81-FD 456 7.067458 0.8540.823 0.854 0.8437 0.8524 0.889 0.8917 0.9075 0.9111 026_355_00285180Brain 355 U-118 MG 456 7.408088 0.965 1.005 0.945 0.9352 0.9307 0.9320.9407 0.945 0.9851 026_347_00282740 Brain 347 T98G 456 7.802123 0.9861.098 0.951 1.0602 0.9957 1.038 1.0462 0.9753 0.982 026_417_00271110Breast 417 DU4475 456 −3.0044 0.119 0.118 0.124 0.1202 0.1235 0.1210.164 0.4789 0.6922 26_465_00271670 Breast 465 MRK-nu-1 456 1.7447420.115 0.398 0.572 0.6893 0.7807 0.868 0.921 0.9463 1.1175026_438_00273540 Breast 438 HCC1599 456 1.968855 0.352 0.459 0.6170.7517 0.8021 0.899 0.9102 0.9757 1.0216 026_435_00271290 Breast 435HCC1187 456 2.132259 0.375 0.482 0.801 0.7466 0.7917 0.794 0.9164 0.93520.9707 026_403_00271400 Breast 403 MCF7 456 2.857648 0.493 0.634 0.7650.8761 0.9706 0.973 1.0249 1.0247 1.0456 026_401_00273450 Breast 401MDA-MB-468 456 3.05753 0.415 0.718 0.931 0.9526 0.909 0.954 0.95121.0282 0.9601 26_451_00271640 Breast 451 CAL-85-1 456 3.06898 0.4890.695 0.808 0.7646 0.8147 0.885 0.8459 1.0885 0.9906 026_404_00273430Breast 404 MDA-MB-231 456 3.086092 0.483 0.731 0.783 0.824 0.8358 0.8740.9446 0.9609 1.0599 026_418_00271550 Breast 418 Hs 578T 456 3.1259560.071 0.614 0.832 0.7916 0.8611 0.836 0.9569 0.9556 1.0186026_402_00272120 Breast 402 CAMA-1 456 3.166443 0.55 0.636 0.773 0.81520.9017 0.886 0.906 0.9417 0.9695 026_426_00274200 Breast 426 HCC1569 4563.228782 0.571 0.713 0.945 0.9056 0.9414 0.938 0.9814 1.028 1.0046026_431_00271130 Breast 431 HCC1806 456 3.337319 0.566 0.672 0.68 0.68520.6987 0.765 0.8378 1.0081 1.1468 026_414_00271900 Breast 414 AU565 4563.409308 0.533 0.816 0.949 0.9831 0.9841 0.924 0.9729 0.9876 1.1475026_452_00272130 Breast 452 COLO-824 456 3.645131 0.358 0.743 0.8430.9519 0.9643 0.904 0.9021 1.0288 0.9665 026_416_00271360 Breast 416BT-549 456 3.723874 0.659 0.818 0.911 0.9492 0.9446 0.965 0.9707 0.97851.0314 026_8144_00274240 Breast 8144 OCUB-M 456 3.727884 0.44 0.801 0.890.9127 1.0139 1.046 0.9729 0.96 1.0167 026_432_00271960 Breast 432 HCC70456 3.73179 0.605 0.733 0.855 0.8647 0.8214 0.887 0.9582 0.9394 1.1987026_457_00273420 Breast 457 EVSA-T 456 3.968951 0.686 0.879 0.927 0.93850.9505 0.975 0.9713 0.9606 1.0214 026_466_00274370 Breast 466 YMB-1-E456 3.997753 0.654 0.836 0.87 0.9113 0.9082 0.908 0.9623 1.0457 0.9738026_441_00285120 Breast 441 HCC2157 456 3.997874 0.474 1.044 0.8490.8244 0.6122 0.948 1.0739 0.6695 1.0405 026_443_00271990 Breast 443MDA-MB-330 456 4.004084 0.601 0.92 0.94 0.9085 0.9663 0.971 0.98170.9257 1.0607 026_436_00271300 Breast 436 HCC1395 456 4.036641 0.7030.897 0.946 0.9395 1.0136 1.033 1.0207 1.0407 1.1296 026_412_00277190Breast 412 UACC-893 456 4.234383 0.421 0.859 0.854 0.8448 0.9188 0.9990.9512 1.0422 0.9971 026_450_00271390 Breast 450 CAL-51 456 4.3195450.678 0.725 0.783 0.8043 0.8395 0.899 0.9313 0.9642 1.0975026_449_00271380 Breast 449 CAL-148 456 4.389344 0.747 0.676 1.08 0.95710.7093 0.888 0.981 0.8213 1.2196 026_434_00271920 Breast 434 HCC1143 4564.464516 0.726 0.808 0.808 0.822 0.8993 0.923 0.919 0.949 1.1427026_433_00276270 Breast 433 HCC202 456 4.571252 0.686 0.9 0.944 0.85490.8115 0.943 0.972 0.9241 0.9618 026_422_00274230 Breast 422MDA-MB-175-VII 456 4.594595 0.7 0.73 0.74 0.7666 0.8545 0.848 0.91550.9541 0.9487 026_461_00272170 Breast 461 MFM-223 456 4.656681 0.7870.875 0.908 0.9465 0.988 1.047 1.0015 1.1431 1.0159 026_427_00271330Breast 427 MDA-MB-453 456 4.669025 0.795 0.819 0.921 0.9998 0.9757 0.9981.0349 1.0264 1.1651 026_448_00271370 Breast 448 CAL-120 456 4.7796870.744 0.746 0.796 0.8032 0.8728 0.85 0.9852 1.0071 1.0287026_411_00271420 Breast 411 UACC-812 456 5.072094 0.82 0.894 0.8730.9564 0.9958 0.944 0.9931 1.0326 0.9659 026_442_00273480 Breast 442HCC2218 456 5.225292 0.614 0.923 0.963 1.0597 1.042 1.043 1.0714 1.04981.0318 026_398_00272150 Breast 398 HCC1428 456 5.256241 0.85 0.899 1.0130.8915 0.9851 0.983 0.9959 0.9937 1.0331 026_464_00308490 Breast 464T47D 456 5.26439 0.825 0.853 0.869 0.8588 0.823 0.978 0.9397 0.95620.968 026_400_00273440 Breast 400 MDA-MB-436 456 5.286367 0.725 0.8440.848 0.8326 0.8721 0.851 0.8881 0.9805 0.933 026_437_00280161 Breast437 HCC1500 456 5.288806 0.796 0.982 0.913 0.8847 0.9397 1.088 0.85230.9109 0.9883 026_440_00271950 Breast 440 HCC1954 456 5.303093 0.8070.813 0.885 0.8155 0.8463 0.861 1.0199 1.0209 0.9481 026_413_00271930Breast 413 HCC1419 456 5.337084 0.839 0.926 0.932 0.9634 0.9633 0.9410.9595 0.9516 1.1518 026_410_00272180 Breast 410 ZR-75-30 456 5.3730810.83 0.857 0.88 0.8529 0.9183 0.913 0.911 1.03 0.9685 026_439_00271940Breast 439 HCC1937 456 5.44243 0.841 0.952 0.975 1.0253 0.9632 0.9531.0302 1.0262 1.0774 026_408_00271350 Breast 408 BT-20 456 5.7358720.871 0.955 0.93 0.9913 0.9764 0.972 1.0674 0.9818 1.1818026_399_00272160 Breast 399 MDA-MB-415 456 5.879088 0.884 0.875 0.8850.8851 0.9175 0.914 0.9179 0.9049 0.9735 026_458_00274220 Breast 458HDQ-P1 456 6.259493 0.84 1.072 1.136 1.0763 1.0797 1.042 1.0437 1.02311.0073 026_397_00274350 Breast 397 HCC38 456 6.744219 0.874 1.053 1.0231.0222 0.9856 0.93 0.9274 0.9402 0.9941 026_405_00274360 Breast 405MDA-MB-361 456 6.792889 0.935 0.959 0.987 0.9819 0.9119 0.921 0.95970.998 0.9922 026_425_00280231 Breast 425 MDA-MB-157 456 7.000504 0.9061.069 1.05 0.9597 0.9229 0.966 0.9422 0.9712 0.9545 026_454_00272140Breast 454 EFM-192A 456 7.097342 0.942 1.032 1.041 1.0545 0.9812 0.9791.0186 1.0191 0.9742 026_420_00271541 Breast 420 BT-474 456 7.4589140.191 1.089 1.053 1.0824 1.0008 0.955 0.9764 0.9678 1.0149026_453_00273410 Breast 453 EFM-19 456 8.207256 1.096 1.286 1.208 1.11121.0544 1.042 1.0064 1.0474 1.0572 026_415_00316440 Breast 415 BT-483 4568.21654 1.148 1.275 1.181 1.1462 1.089 1.147 1.0671 1.0911 1.1123026_8176_00316650 Cervix 8176 TC-YIK 456 0.842618 0.29 0.416 0.4880.5211 0.5993 0.669 0.7672 0.7877 1.0572 026_479_00264920 Cervix 479HT-3 456 1.420025 0.257 0.608 0.574 0.5844 0.5901 0.637 0.8649 0.81890.8617 026_478_00271910 Cervix 478 C-33 A 456 2.72591 0.472 0.615 0.6580.6776 0.6753 0.734 0.7964 0.8383 0.9143 026_478_00269410 Cervix 478C-33 A 456 2.976483 0.445 0.701 0.714 0.731 0.8172 0.771 0.9038 0.8520.9634 026_493_00268830 Cervix 493 ME-180 456 3.07379 0.509 0.667 0.6950.7355 0.6828 0.732 0.7953 0.9097 1.0456 026_476_00269050 Cervix 476 C-4I 456 3.232632 0.508 0.792 0.857 0.8645 0.8671 0.933 1.0078 1.04841.1636 026_8145_00271140 Cervix 8145 OMC-1 456 3.295968 0.531 0.786 0.840.8546 0.9307 0.911 1.0261 1.0441 1.0392 026_484_00263710 Cervix 484 CaSki 456 3.639931 0.327 0.715 0.879 0.8811 0.8284 0.817 0.9599 1.03611.165 026_469_00264610 Cervix 469 HeLa 456 3.981495 0.311 0.783 0.9330.9784 0.9807 0.982 0.9321 0.9429 0.9946 026_493_00262480 Cervix 493ME-180 456 4.007923 0.639 0.799 0.885 0.9474 0.9337 0.873 0.9457 0.86940.8949 026_474_00269100 Cervix 474 SiHa 456 4.596545 0.742 0.846 0.8320.8807 0.9276 0.898 1.0004 0.9967 1.1646 026_482_00262520 Cervix 482SISO 456 5.313141 0.851 0.946 0.96 0.9752 0.9888 0.276 0.975 0.9470.9669 026_482_00274250 Cervix 482 SISO 456 5.375837 0.842 0.881 0.8870.9541 0.9605 0.96 0.9679 1.044 1.0481 026_482_00269740 Cervix 482 SISO456 5.709731 0.838 0.89 0.925 0.9202 0.9445 0.931 0.9609 0.9695 0.9938026_468_00264600 Cervix 468 DoTc2 4510 456 5.749229 0.363 0.92 0.9610.9796 0.9907 0.984 1.01 0.9525 0.9858 026_473_00264650 Cervix 473 SW756456 5.953892 0.162 0.868 0.813 0.7938 0.8104 0.779 0.9163 0.8951 0.9568026_491_00264830 Cervix 491 SKG-IIIa 456 6.261878 0.721 1.002 1.0081.007 1.0125 0.97 1.0049 0.9721 0.9931 026_476_00264900 Cervix 476 C-4 I456 6.792994 0.225 0.991 0.994 0.9083 0.8797 0.89 0.982 0.9123 0.9144026_474_00264930 Cervix 474 SiHa 456 7.505839 0.532 0.824 0.865 0.90920.9029 0.879 0.8904 0.8746 0.8785 026_472_00264630 Cervix 472 MS751 4567.679336 1.023 0.945 0.949 0.9806 0.9744 0.979 0.9915 0.9775 0.9824026_8180_00276230 Esophagus 8180 TE-15 456 0.823778 0.313 0.365 0.4480.4963 0.618 0.64 0.8902 0.6859 1.0361 026_502_00276550 Esophagus 502KYSE-450 456 1.219587 0.422 0.41 0.448 0.5484 0.5334 0.703 0.7332 0.88920.853 026_497_00274050 Esophagus 497 KYSE-150 456 1.304746 0.328 0.4020.467 0.567 0.6585 0.801 0.8957 0.9976 0.9719 026_8252_00276570Esophagus 8252 OACp4C 456 1.678468 0.101 0.3 0.567 0.6503 0.7826 0.8610.8531 0.9243 0.9446 026_496_00276530 Esophagus 496 KYSE-140 4562.209734 0.522 0.516 0.476 0.5824 0.6741 0.815 0.8515 0.806 0.8896026_8233_00278570 Esophagus 8233 ESO26 456 2.595405 0.493 0.568 0.6790.7293 0.7791 0.852 0.8801 1.0449 0.8251 026_8184_00282680 Esophagus8184 TE-6 456 2.946928 1.152 0.677 0.704 0.6755 0.7901 0.89 1.19891.0724 0.9825 026_8277_00276670 Esophagus 8277 TE-4 456 3.072573 0.5180.605 0.768 0.8018 0.8549 0.883 0.9093 0.8952 0.9579 026_506_00277170Esophagus 506 OE19 456 3.143883 0.544 0.752 0.743 0.8799 1.0437 0.9371.0332 1.0004 1.0701 026_8179_00276220 Esophagus 8179 TE-12 456 3.1636170.183 0.742 0.633 0.6476 0.7356 0.839 0.7964 0.8189 0.8318026_8185_00276250 Esophagus 8185 TE-8 456 3.50176 0.201 0.606 0.6580.7374 0.6587 0.744 0.775 0.681 0.9258 026_8184_00293680 Esophagus 8184TE-6 456 3.545508 0.552 0.767 0.935 0.8745 0.8626 0.742 1.0239 0.8630.9232 026_8178_00280260 Esophagus 8178 TE-10 456 3.804003 0.664 0.7350.78 0.9124 0.8781 1.01 1.0331 0.9777 1.0127 026_499_00276630 Esophagus499 KYSE-270 456 4.027681 0.678 0.727 0.767 0.7928 0.8774 0.984 0.99111.0386 1.0007 026_509_00276620 Esophagus 509 KYSE-220 456 4.075564 0.6370.656 0.682 0.7368 0.7969 0.844 0.873 0.9181 0.9229 026_8235_00276520Esophagus 8235 FLO-1 456 4.100409 0.604 0.732 0.778 0.7882 0.8071 0.8640.8729 0.8842 0.9483 026_8251_00276640 Esophagus 8251 OACM5-1 4564.181038 0.673 0.784 0.878 0.8883 0.9366 0.926 0.9214 0.9309 0.9714026_8186_00282690 Esophagus 8186 TE-9 456 4.291684 0.689 0.795 0.7420.8146 0.856 0.846 1.0123 0.8588 1.0654 026_495_00274190 Esophagus 495COLO-680N 456 4.332573 0.742 0.818 0.865 0.9122 0.97 0.96 1.1262 0.96841.0444 026_510_00273560 Esophagus 510 KYSE-50 456 4.431288 0.658 0.7430.781 0.8044 0.8395 0.815 0.8665 0.9009 1.0802 026_8186_00292740Esophagus 8186 TE-9 456 4.438695 0.739 0.769 0.893 0.8667 0.8573 0.9441.0623 1.0025 1.1458 026_503_00274070 Esophagus 503 KYSE-510 4564.484808 0.731 0.796 0.816 0.8942 0.9487 0.912 0.9785 1.0104 1.0221026_504_00276560 Esophagus 504 KYSE-520 456 4.773857 0.716 0.731 0.7480.7768 0.8124 0.867 0.9459 0.995 0.9576 026_8208_00276600 Esophagus 8208HCE-4 456 4.88732 0.748 0.785 0.808 0.8645 0.9033 0.954 0.9197 0.92310.9878 026_512_00274080 Esophagus 512 T.T 456 4.943912 0.781 0.839 0.890.9002 0.9769 0.929 0.9845 0.9744 1.0152 026_8268_00276650 Esophagus8268 SK-GT-4 456 5.079273 0.835 0.788 0.758 0.735 0.7503 0.833 0.90630.9163 0.972 026_505_00274210 Esophagus 505 KYSE-70 456 5.137973 0.7940.857 0.892 0.8943 0.9411 0.957 0.9493 0.9907 1.0431 026_508_00278520Esophagus 508 OE33 456 5.424479 0.701 0.81 0.783 0.7873 0.7763 0.8330.8596 0.903 0.9287 026_498_00276540 Esophagus 498 KYSE-180 456 5.5336520.84 0.853 0.898 0.9109 0.9353 0.924 0.9336 0.9199 0.942026_8202_00276660 Esophagus 8202 TE-11 456 5.740526 0.822 0.845 0.8480.8684 0.8821 0.913 0.9297 0.9627 1.0605 026_8039_00276580 Esophagus8039 EC-GI-10 456 5.844841 0.878 0.905 0.892 0.951 0.9277 0.926 0.95551.04 1.0068 026_501_00274060 Esophagus 501 KYSE-410 456 6.135532 0.7620.836 0.846 0.8398 0.8691 0.887 0.9278 0.9729 0.9854 026_8246_00276610Esophagus 8246 KYAE-1 456 6.1525 0.887 0.93 0.92 0.9326 0.9185 0.9780.97 0.9663 0.9315 026_507_00278510 Esophagus 507 OE21 456 6.23551 0.9220.838 0.875 0.7771 0.8079 0.985 0.9753 0.9667 0.9919 026_8183_00276240Esophagus 8183 TE-5 456 6.777236 0.111 1.028 0.936 1.0341 1.0898 0.8760.8843 0.9699 0.8561 026_8177_00282670 Esophagus 8177 TE-1 456 7.8487111.022 1.1 0.912 1.0973 1.0839 1.065 1.0874 1.0775 1.0749026_545_00260020 Head & Neck 545 DOK 456 −0.22061 0.308 0.451 0.330.4014 0.4436 0.534 0.6634 0.7589 0.8693 026_1217_00255750 Head & Neck1217 H3118 456 0.314816 0.254 0.413 0.453 0.4692 0.5199 0.565 0.6910.6788 0.7738 026_526_00308740 Head & Neck 526 PCI-4B 456 1.887412 0.4180.409 0.489 0.7595 0.7429 0.873 0.9067 0.918 1.0403 026_530_00260620Head & Neck 530 PCI-30 456 1.98819 0.142 0.356 0.628 0.7119 0.7851 0.8250.9139 0.8747 0.8098 026_552_00252890 Head & Neck 552 SAT 456 2.5342790.382 0.613 0.727 0.8554 0.7778 0.977 0.8474 0.7957 0.9605026_550_00258980 Head & Neck 550 SCC-4 456 2.544916 0.464 0.564 0.9040.7519 0.7827 0.709 0.7715 0.8596 0.9574 026_1224_00256200 Head & Neck1224 SCC-9 456 2.68588 0.466 0.633 0.641 0.6635 0.6231 0.758 0.82870.8854 0.9263 026_1223_00259190 Head & Neck 1223 SCC-25 456 2.9347930.193 0.664 0.718 0.8957 0.937 0.932 1.0111 0.988 1.0055026_548_00261030 Head & Neck 548 RPMI 2650 456 3.095229 0.489 0.6960.761 0.8664 0.8685 0.911 0.9177 0.9218 0.9624 026_517_00308680 Head &Neck 517 JHU-011 456 3.112112 0.536 0.616 0.696 0.7845 0.8433 0.8130.9157 0.9495 1.0827 026_8011_00257080 Head & Neck 8011 BB30-HNC 4563.217966 0.541 0.677 0.734 0.7445 0.7909 0.917 0.9622 0.9832 1.0203026_553_00259140 Head & Neck 553 OSC-20 456 3.378306 0.587 0.751 0.7920.8761 0.9722 0.983 1.0097 0.974 0.999 026_556_00257220 Head & Neck 556SKN-3 456 3.392608 0.207 0.626 0.805 0.7174 0.6353 0.718 0.757 0.85930.8383 026_536_00256080 Head & Neck 536 BHY 456 3.443799 0.593 0.8210.877 0.8931 0.9405 1.049 1.0289 0.9991 1.0281 026_561_00257110 Head &Neck 561 Ca9-22 456 3.710495 0.634 0.867 0.849 0.916 0.9493 0.99 1.01551.0225 1.0222 026_532_00308750 Head & Neck 532 PCI-6A 456 3.742916 0.6280.684 0.811 0.8452 0.8862 0.945 0.9298 0.9056 0.9974 026_8012_00266550Head & Neck 8012 BB49-HNC 456 3.77782 0.46 0.775 0.9 0.8967 0.924 0.9680.9717 1.0116 0.9946 026_8100_00256170 Head & Neck 8100 LB771-HNC 4563.926882 0.701 0.899 0.929 0.9987 0.9867 0.989 1.0145 0.9621 0.9588026_1222_00253030 Head & Neck 1222 SCC-15 456 4.189701 0.411 0.772 0.8550.8291 0.7747 1.02 0.8664 0.932 0.9717 026_533_00260900 Head & Neck 533PCI-15A 456 4.236361 1.325 1.15 0.832 0.8691 0.8188 0.863 0.9549 1.04271.0471 026_547_00314270 Head & Neck 547 KOSC-2 cl3-43 456 4.563275 0.7080.872 0.897 0.9 0.7378 0.941 1.0131 0.9825 0.9292 026_544_00256140 Head& Neck 544 Detriot 562 456 4.601961 0.746 0.964 0.951 0.9801 0.96560.961 0.9765 0.9793 0.9061 026_543_00256100 Head & Neck 543 BICR 78 4564.851894 0.728 0.818 0.868 0.8349 0.9283 0.925 0.9696 0.966 0.9649026_537_00256120 Head & Neck 537 CAL-33 456 5.580216 0.834 0.892 0.9210.9097 0.9511 0.935 0.9977 1.0062 0.9837 026_549_00256220 Head & Neck549 HO-1-N-1 456 5.63511 0.866 0.873 0.883 0.8726 0.8907 0.97 0.9870.9898 1.0714 026_557_00259180 Head & Neck 557 SAS 456 6.035072 0.9130.97 0.967 0.9738 0.9866 1.043 1.0896 1.0407 1.0277 026_534_00256110Head & Neck 534 CAL 27 456 6.045151 0.85 0.904 0.912 0.9312 0.925 0.9390.9758 0.9901 0.9927 026_542_00269190 Head & Neck 542 BICR 31 4566.045414 0.903 0.908 0.952 0.9408 0.977 0.97 0.9966 1.0002 1.0269026_530_00262500 Head & Neck 530 PCI-30 456 6.119674 0.884 0.955 1.1450.9682 1.009 0.988 0.9261 1.0121 0.9861 026_540_00258490 Head & Neck 540BICR 10 456 6.128463 0.839 1.048 1.049 1.0556 1.0464 1.041 1.0301 0.9740.9953 026_541_00256090 Head & Neck 541 BICR 22 456 6.156446 0.828 1.0761.058 0.9602 1.0898 1.064 1.0874 0.9689 1.0462 026_8003_00263440 Head &Neck 8003 A253 456 6.291179 0.877 0.857 0.859 0.8594 0.9753 0.982 0.99850.9367 0.969 026_535_00256160 Head & Neck 535 FaDu 456 6.303565 0.8411.063 1.091 1.0795 1.0793 1.034 1.0404 1.0191 1.0095 026_554_00257210Head & Neck 554 OSC-19 456 6.322489 0.938 0.942 0.914 0.9247 0.96821.004 1.0285 1.0504 1.0506 026_559_00256250 Head & Neck 559 HSC-3 4566.797258 0.909 1.024 1.057 1.0632 1.0572 1.029 1.0059 0.9701 0.9782026_8071_00256760 Head & Neck 8071 HCE-T 456 6.917353 0.576 1.146 1.2741.3514 1.1688 1.173 1.0336 1.1999 0.8849 026_521_00257180 Head & Neck521 JHU-022 456 7.049785 0.896 0.846 0.901 0.8757 0.9278 0.886 0.91030.9436 0.9699 026_555_00256270 Head & Neck 555 KON 456 7.114386 0.9460.982 0.977 0.9826 0.9957 0.96 0.9991 0.961 0.9207 026_538_00258510 Head& Neck 538 HN 456 7.118392 0.947 1.007 0.984 1.0014 1.0052 0.998 1.01050.9648 0.9952 026_546_00259150 Head & Neck 546 PE/CA-PJ15 456 7.262190.96 0.963 0.931 0.9179 0.9698 0.969 0.9884 1.006 0.9974026_560_00256260 Head & Neck 560 HSC-4 456 7.41962 1.005 0.994 1.0030.9486 1.2114 0.897 0.9756 0.9433 0.9255 026_551_00259130 Head & Neck551 HO-1-u-1 456 7.903602 0.999 1.037 1.039 1.0722 1.0616 1.067 1.06551.0038 0.9405 026_558_00256240 Head & Neck 558 HSC-2 456 8.185707 1.1481.077 0.985 1.1269 1.0034 1.093 0.9608 0.9252 0.9888 026_531_00258970Head & Neck 531 PCI-38 456 8.493869 1.046 1.067 1.063 1.0751 1.07120.965 1.0236 1.0421 0.9848 026_570_00293670 Intestine 570 SK-CO-1 456−0.30187 0.268 0.258 0.267 0.2555 0.3743 0.418 0.7306 0.9824 0.9276026_8153_00295901 Intestine 8153 RKO 456 0.044041 0.222 0.197 0.2460.3161 0.4612 0.693 0.7818 0.9061 1.1131 18 Intestine 586 COLO 205 4560.350012 0.083 0.182 0.268 0.3978 0.5206 0.706 0.8518 0.9472 0.9745026_582_00295550 Intestine 582 LoVo 456 0.399717 0.325 0.396 0.3850.4168 0.4097 0.533 0.75 0.8239 0.9276 026_8108_00298530 Intestine 8108LS-513 456 0.423432 0.094 0.146 0.195 0.2527 0.5673 0.876 1.1755 1.01791.1926 026_8274_00258540 Intestine 8274 SNU-61 456 0.467862 0.432 0.5990.539 0.5369 0.5735 0.603 0.7283 0.7462 0.9243 026_8136_00260060Intestine 8136 NCI-H747 456 0.578701 0.356 0.465 0.488 0.5338 0.54710.613 0.7238 0.8107 0.9473 026_574_00298390 Intestine 574 CL-11 4560.894127 0.381 0.401 0.395 0.4593 0.6002 0.69 0.749 0.8474 0.9789026_589_00295371 Intestine 589 HCT 116 456 0.931657 0.336 0.381 0.4350.4732 0.5791 0.862 1.1049 1.0627 1.1072 026_608_00293620 Intestine 608CCK-81 456 1.046144 0.242 0.394 0.399 0.4912 0.6325 0.738 0.9653 0.90690.9123 026_610_00293660 Intestine 610 RCM-1 456 1.475794 0.45 0.4160.414 0.6252 0.6775 0.804 0.981 1.0277 1.0565 026_569_00295390 Intestine569 HT-29 456 1.526342 0.48 0.464 0.5 0.5547 0.5893 0.74 0.9536 0.90480.9241 026_8271_00314300 Intestine 8271 SNU-175 456 1.739925 0.382 0.5220.535 0.5651 0.6481 0.701 0.7957 0.9458 0.9851 026_8108_00296610Intestine 8108 LS-513 456 1.815428 0.37 0.448 0.511 0.6156 0.8074 0.9330.9905 0.9579 0.9991 026_592_00295380 Intestine 592 HT115 456 1.8696890.377 0.436 0.5 0.6258 0.8213 1.101 1.0858 1.0169 1.1148026_606_00293630 Intestine 606 HCC-56 456 1.929923 0.646 0.514 0.5360.5915 0.7207 0.867 0.8829 0.9131 0.9417 026_8107_00296450 Intestine8107 LS-411N 456 2.297731 0.497 0.55 0.585 0.5585 0.6884 0.755 0.86260.8921 0.9253 026_595_00295420 Intestine 595 LS180 456 2.32965 0.4530.515 0.551 0.6999 0.8666 0.91 1.0102 0.9178 1.1155 026_8169_00295910Intestine 8169 SNU-C2B 456 2.617884 0.503 0.566 0.598 0.7373 0.671 0.7740.9066 0.8796 0.9886 026_564_00292860 Intestine 564 NCI-H630 4562.627492 0.496 0.556 0.55 0.7713 1.0007 0.964 0.9563 0.8783 0.9169026_603_00292731 Intestine 603 SW837 456 2.797981 0.525 0.597 0.5860.7811 0.7763 1.002 1.023 1.0218 1.0696 026_588_00295360 Intestine 588GP5d 456 2.925873 0.521 0.65 0.67 0.679 0.8325 0.964 1.0479 1.10131.0678 026_598_00302650 Intestine 598 SW 1417 456 3.077195 0.616 0.6960.742 0.6531 1.034 1.029 1.0798 0.8321 1.2207 026_8276_00296000Intestine 8276 SNU-C5 456 3.153347 0.603 0.592 0.612 0.6327 0.7283 0.8120.8824 0.9411 0.9768 026_593_00295530 Intestine 593 HT55 456 3.1641860.589 0.646 0.61 0.7857 0.7974 1.03 1.1134 1.0941 1.1175026_8106_00264670 Intestine 8106 LS-123 456 3.191118 0.547 0.687 0.7830.7554 0.8823 0.879 0.9202 0.965 0.9375 026_599_00296340 Intestine 599SW 1463 456 3.237942 0.572 0.642 0.631 0.6255 0.6275 0.757 0.9237 0.94010.9681 026_8086_00296431 Intestine 8086 KM12 456 3.240654 0.564 0.6570.65 0.6739 0.8062 0.903 0.8847 0.9825 0.9793 026_587_00302320 Intestine587 COLO 741 456 3.269221 0.615 0.622 0.694 0.6624 0.7236 0.816 1.10471.0995 1.0783 026_8273_00295970 Intestine 8273 SNU-407 456 3.3904030.577 0.622 0.663 0.7325 0.7399 0.745 0.8059 0.8644 1.0342026_8270_00304630 Intestine 8270 SNU-1040 456 3.643993 0.604 0.78 0.7990.8256 0.8028 0.92 0.9246 0.9919 1.2066 026_8168_00295990 Intestine 8168SNU-C1 456 3.717495 0.645 0.673 0.685 0.6875 0.8368 0.739 0.8513 1.11151.0883 026_8275_00256210 Intestine 8275 SNU-81 456 3.903109 0.338 0.6590.68 0.6942 0.7065 0.761 0.7749 0.8653 0.9224 026_583_00295921 Intestine583 SW-948 456 3.908038 0.735 0.672 0.851 0.9918 1.0392 0.979 0.91780.9736 0.9758 026_8274_00295980 Intestine 8274 SNU-61 456 4.14154 0.6630.72 0.672 0.6639 0.7521 0.794 0.8775 0.8761 0.9371 026_8105_00296440Intestine 8105 LS-1034 456 4.222062 0.732 0.754 0.781 0.8085 0.88571.006 1.1032 1.0973 1.0698 026_580_00295930 Intestine 580 COLO-678 4564.449849 0.891 0.793 0.767 0.8394 0.8741 0.917 1.0326 0.9344 1.0884026_580_00266560 Intestine 580 COLO-678 456 4.550688 0.724 0.686 0.7850.8147 0.8701 0.885 0.9546 0.9558 0.9803 026_581_00295830 Intestine 581HCT-15 456 4.608693 0.811 0.75 0.973 0.9405 1.0972 0.98 1.098 1.07221.1405 026_573_00296370 Intestine 573 SW620 456 4.816766 0.775 0.7870.797 0.8519 0.8683 0.915 0.9626 1.0901 1.097 026_8021_00296390Intestine 8021 COLO-320-HSR 456 4.87923 0.817 0.861 0.828 1.0219 0.89741.103 1.0927 0.9967 1.1145 026_8106_00298521 Intestine 8106 LS-123 4564.895207 0.706 0.749 0.852 0.7598 0.7605 0.923 0.8934 0.9319 1.0175026_600_00296361 Intestine 600 SW 48 456 4.951306 0.706 0.776 0.7750.7937 0.8445 0.857 0.9382 0.915 0.9609 026_8070_00296411 Intestine 8070HCC2998 456 4.984157 0.701 0.788 0.743 0.7463 0.8151 0.891 0.9115 0.95720.9432 026_8135_00295950 Intestine 8135 NCI-H716 456 4.988916 0.8130.864 0.948 0.9832 0.9658 0.94 0.9372 0.9633 1.1237 026_8136_00295961Intestine 8136 NCI-H747 456 5.220086 0.699 0.693 0.763 0.7315 0.78630.815 0.8715 0.8842 1.1461 026_574_00263900 Intestine 574 CL-11 4565.294016 0.826 0.812 0.761 0.7543 0.8087 0.891 0.9784 0.9577 0.9649026_8026_00300671 Intestine 8026 CW-2 456 5.403672 0.827 0.955 0.9740.9551 0.9288 0.954 0.9824 0.9866 1.0636 026_607_00293610 Intestine 607CaR-1 456 5.551737 0.834 0.873 0.912 0.8795 0.9318 0.939 0.9636 0.94830.9415 026_8074_00296420 Intestine 8074 HUTU-80 456 5.701754 0.841 0.8520.876 0.9027 0.8952 1.007 1.0026 0.966 0.9322 026_563_00316540 Intestine563 C2BBe1 456 5.783056 0.853 0.949 0.926 0.9333 0.9253 0.879 0.91641.0369 1.0733 026_596_00296280 Intestine 596 MDST8 456 6.402689 0.8850.885 0.874 0.8768 0.9374 0.931 0.9758 0.9779 1.0734 026_597_00300651Intestine 597 SW 1116 456 6.447765 0.647 1.058 0.925 1.031 0.9839 0.9971.0631 1.0277 0.905 026_601_00296480 Intestine 601 T84 456 7.2336310.973 0.939 0.93 0.9736 0.9545 0.891 0.964 0.9372 1.0259026_622_00288160 Kidney 622 G-401 456 1.158385 0.363 0.358 0.433 0.50450.6202 0.865 0.8778 1.0261 1.0061 026_626_00298790 Kidney 626 BFTC-909456 1.589633 0.292 0.387 0.526 0.5522 0.8154 0.895 0.9347 1.005 1.054026_623_00288201 Kidney 623 SK-NEP-1 456 1.837955 0.412 0.444 0.5070.7421 0.8141 0.813 0.9099 0.9209 0.981 026_8264_00290630 Kidney 8264RCC-JF 456 2.377362 0.452 0.535 0.585 0.6574 0.9675 0.851 0.9828 1.05911.0712 026_619_00290290 Kidney 619 769-P 456 2.463867 0.439 0.58 0.5760.7655 0.8898 0.994 1.0776 1.0508 1.1513 026_627_00291130 Kidney 627CAL-54 456 2.931844 0.586 0.588 0.649 0.6813 0.7826 0.858 0.9811 0.97261.0003 026_617_00290310 Kidney 617 ACHN 456 2.983353 0.545 0.592 0.6270.7345 0.7447 0.733 0.7712 0.919 1.1152 026_8263_00290620 Kidney 8263RCC-FG2 456 3.111338 0.517 0.689 0.691 0.7857 0.7896 0.884 0.8746 0.97541.0284 026_8190_00290280 Kidney 8190 TK10 456 3.318654 0.638 0.654 0.6710.7606 0.892 0.932 1.0085 1.0009 0.9983 026_638_00288220 Kidney 638VMRC-RCZ 456 3.36569 0.14 0.709 0.838 0.9211 0.92 0.981 0.9922 0.95880.989 026_8261_00308760 Kidney 8261 RCC-AB 456 3.394556 0.566 0.7240.758 0.85 0.8258 0.948 0.9492 0.9471 0.9351 026_628_00288210 Kidney 628SW 13 456 3.41636 0.454 0.733 0.735 0.7572 0.8068 0.826 0.8584 0.93611.0195 026_8262_00290610 Kidney 8262 RCC-ER 456 3.745967 0.583 0.7830.794 0.8415 0.9082 0.87 0.8861 0.9471 0.9855 026_8265_00302360 Kidney8265 RCC-JW 456 3.749626 0.686 0.758 0.874 0.9595 1.0107 1.003 1.01351.0068 1.0345 026_8261_00311200 Kidney 8261 RCC-AB 456 3.919417 0.2880.72 0.73 0.765 0.8515 0.878 0.9287 0.8965 0.9174 026_618_00290300Kidney 618 786-O 456 3.967789 0.657 0.719 0.822 0.8016 0.8829 0.8981.0239 0.917 1.0472 026_625_00290670 Kidney 625 UO-31 456 3.998088 0.6620.756 0.767 0.9002 0.8304 0.876 0.9012 1.0588 1.1507 026_8096_00290860Kidney 8096 LB2241-RCC 456 3.99857 0.643 0.686 0.74 0.8137 0.8533 0.9020.9235 0.9107 0.9322 026_614_00291210 Kidney 614 SW 156 456 4.1372430.651 0.665 0.71 0.7683 0.8365 0.896 0.903 0.9074 0.9013026_8249_00295560 Kidney 8249 NCC021 456 4.201277 0.701 0.897 0.9030.9199 0.9735 0.942 0.9933 1.0276 1.0113 026_633_00290250 Kidney 633KMRC-20 456 4.24311 0.713 0.872 0.83 0.9644 0.8737 1.135 1.027 1.09231.0373 026_8068_00290231 Kidney 8068 HA7-RCC 456 4.403879 0.761 0.7640.7 0.8322 0.7944 0.89 1.0474 1.1224 1.0563 026_8095_00290260 Kidney8095 LB1047-RCC 456 4.460236 0.772 0.824 0.914 1.0356 1.0154 1.0041.0739 1.1192 1.1745 026_626_00258890 Kidney 626 BFTC-909 456 4.4642050.741 0.932 1.057 1.0395 1.0888 1.03 1.0217 1.0625 1.0049026_8147_00290270 Kidney 8147 OS-RC-2 456 4.480976 0.767 0.794 0.7990.8302 0.9419 1.023 1.0718 1.0847 1.1694 026_8013_00290220 Kidney 8013BB65-RCC 456 4.50555 0.714 0.769 0.765 0.8909 0.8509 0.865 0.9509 1.00960.903 026_8006_00293300 Kidney 8006 A704 456 4.630889 0.755 0.917 0.9180.9328 0.9788 1.112 0.981 0.9813 1.0413 026_637_00290240 Kidney 637KMRC-1 456 4.786417 0.748 0.889 0.97 0.9322 0.836 1.012 1.0239 0.95031.088 026_624_00290320 Kidney 624 Caki-1 456 4.808296 0.821 0.803 0.9080.9738 0.9722 1.083 1.1676 1.1464 1.1228 026_8266_00290640 Kidney 8266RCC-MF 456 4.811179 0.754 0.903 0.819 0.8455 0.8764 0.999 0.9885 0.9351.1762 026_620_00288170 Kidney 620 G-402 456 4.865533 0.694 0.721 0.6860.7919 0.7766 0.804 0.8404 0.885 0.9093 026_8152_00293381 Kidney 8152RCC10RGB 456 5.110675 0.821 0.719 0.783 0.9032 0.8829 0.858 0.89910.9287 1.0343 026_640_00291220 Kidney 640 VMRC-RCW 456 5.287926 0.730.737 0.793 0.8088 0.8744 0.905 0.9003 0.9127 0.9163 026_8102_00293360Kidney 8102 LB996-RCC 456 5.484542 0.855 0.901 0.954 0.9116 0.9754 0.980.958 0.9786 0.9921 026_1119_00290901 Kidney 1119 SN-12C 456 5.5865870.818 0.749 0.747 0.783 0.8058 0.856 0.9038 0.9508 0.9573026_626_00290810 Kidney 626 BFTC-909 456 5.753321 0.799 0.854 0.9030.9293 0.9289 0.92 0.9333 0.9322 0.9535 026_8005_00266530 Kidney 8005A498 456 5.843211 0.594 0.917 1.009 1.0079 0.9307 0.996 1.0002 1.00330.9941 026_8157_00296471 Kidney 8157 RXF393 456 6.166218 0.853 0.8780.91 0.8924 0.9071 0.932 0.9081 0.9505 0.9889 026_8102_00253000 Kidney8102 LB996-RCC 456 6.306723 0.959 0.873 0.878 0.9741 0.9596 1.025 0.95250.9849 0.9931 026_8006_00263880 Kidney 8006 A704 456 6.969306 1.3510.929 1.157 0.9881 1.0206 0.957 1.0009 0.9583 0.9931 026_8152_00256190Kidney 8152 RCC10RGB 456 7.15535 1.178 0.835 1.76 0.846 0.8683 1.1231.1733 1.1056 1.0876 026_8005_00296380 Kidney 8005 A498 456 8.1907831.022 1.025 1.024 1.0158 1.03 1.032 1.0128 1.0239 1.0104026_233_00277420 Leukemia 233 SIG-M5 456 −5.883853 0.228 0.243 0.2330.2401 0.2362 0.247 0.2641 0.2732 0.4405 026_217_00277380 Leukemia 217OCI-AML2 456 −1.42786 0.09 0.102 0.106 0.1256 0.1044 0.132 0.3277 0.83330.8618 026_179_00314500 Leukemia 179 KMOE-2 456 −0.249088 0.13 0.1350.138 0.1591 0.3658 0.608 0.865 0.8967 1.0615 026_214_00285590 Leukemia214 NB-4 456 −0.011745 0.18 0.323 0.315 0.3611 0.4215 0.592 0.81440.8865 0.9464 026_168_00280410 Leukemia 168 JURL-MK1 456 0.181843 0.1450.179 0.186 0.2526 0.4638 0.727 0.9379 0.904 0.9907 026_194_00280680Leukemia 194 ML-2 456 0.294246 0.418 0.395 0.379 0.4439 0.4739 0.6330.7874 0.8519 1.0223 026_186_00280670 Leukemia 186 LAMA-84 456 0.2959790.152 0.15 0.191 0.3623 0.6147 0.774 0.777 0.8716 0.8816026_234_00314650 Leukemia 234 SKM-1 456 0.349518 0.37 0.347 0.369 0.44390.4995 0.57 0.729 0.9566 1.2103 026_221_00280300 Leukemia 221 OCI-M1 4560.656393 0.183 0.235 0.344 0.5052 0.5942 0.759 0.8267 0.8418 1.1212026_260_00280430 Leukemia 260 KO52 456 0.674413 0.348 0.376 0.357 0.34460.4195 0.753 0.7217 0.7637 1.0996 026_45_00274530 Leukemia 45 HL-60 4560.756274 0.344 0.389 0.403 0.4669 0.6154 0.78 0.9211 0.9579 1.0107026_218_00279140 Leukemia 218 OCI-AML3 456 0.868641 0.368 0.38 0.4010.4571 0.5459 0.76 0.8359 0.9311 0.9403 026_219_00280290 Leukemia 219OCI-AML5 456 0.879404 0.245 0.404 0.427 0.4943 0.686 0.712 0.8112 0.94980.9021 026_199_00314510 Leukemia 199 MOLT-13 456 0.892109 0.227 0.3390.454 0.5097 0.5774 0.766 0.8101 0.9633 1.1416 026_226_00280310 Leukemia226 PL-21 456 0.938066 0.406 0.425 0.463 0.4834 0.537 0.657 0.73380.7978 0.8482 026_8141_00274380 Leukemia 8141 NOMO-1 456 1.040856 0.3750.445 0.435 0.4798 0.5465 0.636 0.9597 0.9214 0.9288 026_8069_00279180Leukemia 8069 HAL-01 456 1.147185 0.349 0.345 0.454 0.634 0.6764 1.1030.7322 0.8261 1.0097 026_68_00273640 Leukemia 68 MV-4-11 456 1.1553220.061 0.367 0.445 0.5488 0.6586 0.777 0.8697 0.9167 1.045026_175_00282940 Leukemia 175 KARPAS-620 456 1.190853 0.562 0.517 0.4440.6051 0.7411 0.916 0.9551 1.1003 1.0193 026_89_00278880 Leukemia 89MEG-01 456 1.211836 0.223 0.376 0.433 0.6062 0.6931 0.86 0.9075 0.99931.0376 026_8017_00279150 Leukemia 8017 BV-173 456 1.464827 0.147 0.4540.57 0.8503 0.5534 0.638 0.7679 0.8629 1.0089 026_225_00277400 Leukemia225 PF-382 456 1.646942 0.317 0.403 0.525 0.6985 0.7396 0.932 0.96110.9018 1.0833 026_285_00282960 Leukemia 285 KY821A3 456 1.656661 0.4220.426 0.445 0.6672 0.7837 0.935 1.0015 1.0293 1.0583 026_8008_00280620Leukemia 8008 ALL-PO 456 1.659142 0.143 0.469 0.549 0.6225 0.6911 0.7250.845 0.8482 1.019 026_177_00276860 Leukemia 177 KE-37 456 1.7114070.195 0.451 0.523 0.6745 0.9068 0.897 0.9087 0.9169 1.0477026_126_00280270 Leukemia 126 GDM-1 456 1.723253 0.297 0.444 0.595 0.6820.7944 0.786 0.9053 0.9081 0.8666 026_261_00274440 Leukemia 261 MY-M12456 1.811821 0.395 0.471 0.59 0.6443 0.7461 0.832 0.8778 1.0023 1.0569026_201_00277390 Leukemia 201 MOLT-16 456 1.821177 0.276 0.43 0.5780.7394 0.7801 0.857 0.9379 1.0322 0.9678 026_148_00277330 Leukemia 148CMK 456 1.894859 0.275 0.484 0.637 0.6101 0.744 0.771 0.9002 0.89490.8668 026_28_00280320 Leukemia 28 SUP-B15 456 1.921651 0.261 0.5060.579 0.7214 0.7165 0.82 0.908 0.9679 0.9913 026_190_00287920 Leukemia190 ME-1 456 1.924633 0.611 0.596 0.587 0.5806 0.5987 0.684 0.79060.8581 0.9106 026_8150_00273700 Leukemia 8150 QIMR-WIL 456 1.9283330.441 0.546 0.595 0.6574 0.7011 0.771 0.852 0.962 0.9148026_8156_00273710 Leukemia 8156 RPMI-8866 456 1.939976 0.339 0.474 0.5980.694 0.7428 0.867 0.9518 0.9936 0.9682 026_161_00277350 Leukemia 161HC-1 456 1.948766 0.447 0.517 0.568 0.6165 0.6342 0.768 0.7642 0.90741.0155 026_195_00279120 Leukemia 195 MOLM-13 456 1.961596 0.394 0.4550.518 0.6868 0.817 0.872 0.9157 0.983 1.0345 026_209_00279130 Leukemia209 NALM-6 456 2.000316 0.14 0.471 0.592 0.6915 0.7587 0.808 0.86580.9205 0.9768 026_127_00278810 Leukemia 127 CESS 456 2.000616 0.3420.505 0.625 0.6869 0.7679 0.843 0.9355 0.9565 0.9827 026_8196_00278790Leukemia 8196 697 456 2.032634 0.337 0.476 0.617 0.7249 0.8167 0.8851.0481 1.0695 1.0723 026_223_00276841 Leukemia 223 P12-ICHIKAWA 4562.094412 0.266 0.472 0.62 1.0139 0.9551 0.864 0.9219 1.0283 0.907026_157_00279291 Leukemia 157 DND-41 456 2.109279 0.354 0.498 0.6240.7174 0.8189 0.89 0.9495 0.9928 1.0144 026_174_00276850 Leukemia 174KARPAS-45 456 2.156973 0.368 0.541 0.63 0.778 0.8566 0.998 1.0142 0.95610.9985 026_223_00274461 Leukemia 223 P12-ICHIKAWA 456 2.170641 0.2740.465 0.567 0.909 0.9914 1.041 1.0272 0.9908 1.0038 026_231_00277410Leukemia 231 RPMI-8402 456 2.171412 0.292 0.525 0.673 0.8379 0.894 0.9561.0525 1.0069 1.0163 026_176_00278840 Leukemia 176 KCL-22 456 2.1784180.342 0.551 0.66 0.7185 0.7521 0.822 0.8942 1.034 0.9817026_198_00279320 Leukemia 198 MOLP-8 456 2.182175 0.422 0.613 0.6040.6789 0.7123 0.753 0.8469 0.8678 0.9555 026_35_00274541 Leukemia 35MOLT-4 456 2.185513 0.368 0.518 0.657 0.7557 0.8266 0.893 0.9057 1.06491.0505 26_256_00273800 Leukemia 256 U266B1 456 2.191112 0.423 0.53 0.5960.6351 0.7316 0.782 0.8701 0.9616 0.9089 026_41_00278850 Leukemia 41KG-1 456 2.260524 0.445 0.507 0.569 0.7321 0.8443 0.89 0.9006 0.92370.9648 026_38_00278900 Leukemia 38 THP-1 456 2.296961 0.389 0.568 0.6920.7667 0.7899 0.895 0.9332 0.9649 0.9902 026_153_00277340 Leukemia 153CTV-1 456 2.301756 0.308 0.563 0.682 0.7882 0.8949 0.972 1.0362 1.02131.0568 26_284_00273770 Leukemia 284 KY821 456 2.31293 0.459 0.524 0.5990.642 0.7677 0.831 0.9119 0.9596 0.9938 026_256_00304780 Leukemia 256U266B1 456 2.374209 0.453 0.543 0.608 0.6629 0.7604 0.806 0.9117 0.95470.9487 026_8033_00279160 Leukemia 8033 DEL 456 2.390133 0.433 0.5950.647 0.7617 0.7648 0.851 0.9096 0.9116 0.9641 026_227_00314640 Leukemia227 RCH-ACV 456 2.405281 0.39 0.571 0.742 0.8517 0.9043 0.946 0.99171.0175 1.1197 026_33_00274521 Leukemia 33 CCRF-CEM 456 2.408403 0.3540.606 0.685 0.7594 0.8191 0.904 0.9597 0.9629 1.0269 026_141_00276820Leukemia 141 K-562 456 2.457405 0.51 0.504 0.624 0.8081 0.8379 1.1630.9404 1.0057 1.0193 026_8137_00273680 Leukemia 8137 NKM-1 456 2.5432630.464 0.601 0.622 0.6486 0.7425 0.801 0.8993 0.9038 0.9449026_36_00274550 Leukemia 36 Reh 456 2.569808 0.418 0.628 0.742 0.83670.88 0.896 0.9262 0.8979 0.9849 026_8042_00285400 Leukemia 8042EoL-1-cell 456 2.64235 0.232 0.569 0.651 0.7409 0.7743 0.894 0.83330.905 0.9094 026_281_00273690 Leukemia 281 P30/OHK 456 2.804566 0.4730.654 0.769 0.9121 0.8865 1.022 1.0746 1.0233 1.0407 026_59_00278820Leukemia 59 J.RT3-T3.5 456 2.830428 0.457 0.659 0.795 0.8429 0.92591.002 1.0587 0.9873 1.1396 026_164_00277360 Leukemia 164 HEL 4562.835925 0.426 0.745 0.939 0.9081 1.0194 0.987 1.0611 1.0159 0.9841026_183_00277370 Leukemia 183 L-363 456 2.864138 0.468 0.685 0.82 0.90250.9548 1.046 1.0249 0.9812 1.02 026_90_00282840 Leukemia 90 KU812 4562.874545 0.446 0.74 0.806 0.917 0.9589 0.978 0.9646 1.0111 1.0409026_27_00278890 Leukemia 27 RS4;11 456 2.914096 0.47 0.684 0.809 0.88950.9144 0.975 0.9547 0.9619 1.0194 026_167_00314490 Leukemia 167 JURKAT456 2.954834 0.503 0.713 0.842 0.9399 0.9544 0.985 0.9987 1.034 1.0702026_181_00278860 Leukemia 181 KOPN-8 456 2.957727 0.484 0.669 0.7310.7843 0.8662 0.92 0.9473 0.9339 0.9555 026_8155_00309110 Leukemia 277RPMI 8226 456 2.959542 0.648 0.63 0.689 0.8376 0.888 0.99 0.9938 1.01821.0157 026_8041_00280640 Leukemia 8041 EM-2 456 3.022652 0.557 0.6870.614 0.634 0.706 0.679 0.8613 0.8617 1.9538 026_142_00279280 Leukemia142 ALL-SIL 456 3.085854 0.187 0.624 0.769 0.7092 0.7975 0.937 0.95250.9717 0.8276 026_277_00280420 Leukemia 277 RPMI 8226 456 3.102844 0.6590.636 0.566 0.6117 0.6594 0.722 0.8693 0.9508 0.9057 026_180_00279310Leukemia 180 KMS-12-BM 456 3.125028 0.512 0.704 0.788 0.8503 0.87030.949 0.905 0.9806 0.9931 026_8066_00279170 Leukemia 8066 GR-ST 4563.192114 0.532 0.681 0.762 0.8318 0.8448 0.865 1.0258 0.9609 0.998926_283_00273780 Leukemia 283 LC4-1 456 3.243599 0.592 0.732 0.916 0.95221.0268 1.078 0.9517 1.0295 1.0135 026_114_00273650 Leukemia 114 SUP-T1456 3.397019 0.637 0.807 0.908 1.0154 0.984 1.031 0.99 0.9877 1.0134026_138_00287901 Leukemia 138 Loucy 456 3.501279 0.289 0.749 0.772 0.9420.9225 0.948 0.9678 0.9653 1.0654 026_8014_00282911 Leukemia 8014 BE-13456 3.615529 0.68 0.757 0.965 1.1246 1.0528 1.075 1.0884 1.0694 1.103726_274_00273860 Leukemia 274 BALL-1 456 3.704228 0.65 0.709 0.777 0.87680.9725 0.983 0.9819 0.9839 0.9978 026_222_00280480 Leukemia 222 OPM-2456 3.757754 0.681 0.619 0.771 0.9443 0.8789 1.073 0.9014 0.9191 0.9051026_8164_00283540 Leukemia 8164 SK-MM-2 456 3.790344 0.669 0.788 0.8610.8903 0.938 0.941 1.0087 0.9277 0.9972 026_166_00278830 Leukemia 166JJN-3 456 3.901981 0.636 0.791 0.85 0.8566 0.8711 0.916 0.9455 0.98921.057 026_230_00282970 Leukemia 230 ROS-50 456 4.130585 0.741 0.7750.805 0.9413 0.9813 1.11 1.1334 1.0559 0.999 026_159_00314480 Leukemia159 EJM 456 4.251735 0.714 0.845 0.899 0.9099 1.0285 0.923 1.0677 1.07681.0543 026_278_00304730 Leukemia 278 KMS-12-PE 456 4.269602 0.765 0.8140.84 1.0641 0.988 1.023 1.0045 1.0284 1.0665 026_8219_00282850 Leukemia8219 Mo-T 456 4.313664 0.73 0.825 0.804 0.9425 0.9233 1.033 0.84541.1551 1.0561 026_279_00274450 Leukemia 279 P31/FUJ 456 4.386897 0.7050.805 0.799 0.819 0.8632 1.043 1.0364 0.9822 1.0363 026_171_00285190Leukemia 171 KARPAS-231 456 4.404825 0.703 0.9 0.906 0.9339 0.9295 0.9040.9725 0.9496 0.9164 026_244_00273660 Leukemia 244 TALL-1 456 4.4110160.698 0.758 0.785 0.8345 0.8593 0.861 1.0445 1.0026 1.0194026_158_00291340 Leukemia 158 EHEB 456 4.509743 0.665 0.745 0.765 0.78410.8282 0.839 0.92 0.9348 1.0344 026_134_00278800 Leukemia 134 ARH-77 4564.545002 0.781 0.856 0.96 0.848 1.0568 1.029 1.0104 1.0156 1.0086026_246_00273670 Leukemia 246 U-698-M 456 4.580953 0.754 0.891 0.9360.9419 0.9607 0.937 1.032 0.9352 1.0229 026_8113_00280440 Leukemia 8113MHH-CALL-2 456 4.648418 0.771 0.883 0.929 0.9704 0.9337 0.987 1.03451.0223 1.0471 026_159_00311690 Leukemia 159 EJM 456 4.699945 0.746 0.8940.921 0.9275 0.9393 0.941 0.9575 0.9338 0.9675 026_159_00282920 Leukemia159 EJM 456 4.832393 0.746 0.976 1.016 0.9859 1.0015 1.002 1.1587 1.10411.0548 026_8115_00280450 Leukemia 8115 MN-60 456 4.861437 0.826 0.8040.822 1.0586 0.9555 0.947 1.0471 1.128 1.0314 026_204_00280460 Leukemia204 MONO-MAC-6 456 4.946039 0.736 0.802 0.771 0.8409 0.8712 0.833 0.86341.0677 0.855 026_8117_00280470 Leukemia 8117 MUTZ-1 456 5.304526 0.8230.984 1.063 1.051 1.0142 1.007 0.9986 0.9654 0.9525 026_188_00282930Leukemia 188 LP-1 456 5.373997 0.831 0.887 0.972 0.8808 0.9105 0.8860.9533 0.9576 1.138 026_8081_00285580 Leukemia 8081 JVM-3 456 5.5813280.884 0.904 0.907 0.9131 1.01 1.097 1.0593 0.9629 1.0333026_159_00309070 Leukemia 159 EJM 456 5.810283 0.882 0.986 0.998 1.09921.0048 1.045 1.0126 1.024 1.0649 026_8080_00280660 Leukemia 8080 JVM-2456 5.982881 0.867 0.876 0.901 0.8865 0.9235 0.909 0.9782 0.8964 0.9789026_278_00306920 Leukemia 278 KMS-12-PE 456 6.629507 0.971 0.92 0.950.9554 0.9046 0.905 0.9657 1.0243 0.9236 026_8010_00280630 Leukemia 8010ATN-1 456 7.14863 1.03 0.958 0.835 0.8896 0.8651 0.907 0.9392 0.85820.9763 26_262_00273790 Leukemia 262 MLMA 456 7.68552 0.485 1.033 1.0771.0216 1.0341 0.962 1.0378 0.9821 1.0245 026_278_00282950 Leukemia 278KMS-12-PE 456 7.74945 0.958 1.101 1.04 1.1177 1.0046 1.029 1.0272 1.03111.0037 026_649_00264910 Liver 649 Hep 3B2.1-7 456 −0.30042 0.134 0.0980.087 0.1121 0.2203 0.601 0.8432 0.8926 0.9089 026_658_00262810 Liver658 JHH-1 456 −0.185127 0.054 0.329 0.515 0.3993 0.5194 0.524 0.66080.6499 0.6863 026_649_00266180 Liver 649 Hep 3B2.1-7 456 −0.037335 0.0730.105 0.093 0.0908 0.1037 0.202 0.8123 0.9207 1.007 026_667_00273550Liver 667 HuH-7 456 1.289162 0.279 0.565 0.539 0.5535 0.7134 0.77 0.86260.9675 0.9662 026_659_00255780 Liver 659 JHH-2 456 1.758427 0.283 0.4730.657 0.7149 0.551 0.819 0.8858 0.8891 1.0202 026_643_00266460 Liver 643SNU-398 456 2.578054 0.415 0.687 0.643 0.6854 0.6879 0.717 0.7896 0.80760.9193 026_667_00269210 Liver 667 HuH-7 456 2.760591 0.501 0.59 0.6650.6852 0.6886 0.781 0.95 1.0287 1.0142 026_647_00269110 Liver 647SNU-387 456 2.930627 0.494 0.772 0.829 0.9402 0.9686 1.046 1.0372 1.02231.2144 026_661_00252500 Liver 661 JHH-7 456 3.286573 0.539 0.889 0.7940.8777 1.0776 0.953 0.9918 1.037 0.9476 026_643_00263980 Liver 643SNU-398 456 3.293137 0.487 0.768 0.854 0.8235 0.8763 0.883 0.9616 1.0681.0718 026_642_00308440 Liver 642 C3A 456 3.554832 0.587 0.68 0.7060.7687 0.817 0.878 0.9697 0.9574 0.9245 026_648_00258350 Liver 648SNU-423 456 3.576003 0.139 0.699 0.902 0.9157 0.9485 0.909 0.9218 0.94691.0028 026_656_00252510 Liver 656 JHH-4 456 3.61979 0.701 0.653 0.6310.8187 0.7968 0.977 0.9161 0.9897 0.9288 026_660_00252490 Liver 660JHH-6 456 3.831185 0.696 0.88 1.034 0.9294 0.9465 1.04 0.9417 1.05280.952 026_644_00252750 Liver 644 SNU-449 456 4.40481 0.664 0.733 0.7640.7954 0.8597 0.896 0.9485 0.9457 0.9162 026_644_00306170 Liver 644SNU-449 456 6.450644 0.96 0.961 0.745 1.0716 1.0197 1.08 0.991 0.90070.9369 26_646_00314060 Liver 646 SNU-475 456 6.501654 0.181 0.97 0.9710.9863 1.0061 1.007 1.0271 1.0202 0.057 026_654_00255800 Liver 654SK-HEP-1 456 6.515068 0.927 0.956 0.961 0.9827 0.9589 0.98 0.9783 0.97260.9998 026_668_00252690 Liver 668 HLE 456 7.25378 0.434 1.006 1.0870.9897 1.1103 0.964 1.006 1.106 0.9541 026_662_00252460 Liver 662 huH-1456 7.722693 0.466 1.165 1.072 1.0401 1.0408 1.136 1.015 1.123 0.9778026_645_00306160 Liver 645 SNU-182 456 7.852049 1.105 1.061 1.063 1.00551.0268 1.053 1.0365 1.0318 1.0842 026_642_00252670 Liver 642 C3A 4567.864415 0.386 1.1 1.007 1.108 0.9463 1.051 0.9803 1.029 0.9218026_830_00304760 Lung 830 NCI-H2135 456 −0.374655 0.298 0.262 0.2460.3049 0.3692 0.528 0.7269 0.8905 0.977 026_698_00300170 Lung 698NCI-H524 456 2.279246 0.443 0.506 0.63 0.8741 0.9302 1.005 1.0131 1.03041.0567 026_672_00314460 Lung 672 NCI-H510A 456 2.367223 0.323 0.5850.763 0.783 0.8906 0.969 0.9972 1.1809 1.0131 026_761_00300410 Lung 761COR-L279 456 3.131703 0.559 0.745 0.868 0.9821 1.0079 1.06 1.0144 1.01821.0709 026_726_00304770 Lung 726 NCI-H2171 456 3.185965 0.524 0.7290.842 0.8655 0.9088 0.871 1.0252 0.9207 1.129 026_740_00302760 Lung 740NCI-H82 456 3.210581 0.542 0.755 0.865 0.9592 1.0522 0.888 0.9137 1.0041.0151 026_787_00302910 Lung 787 SBC-3 456 3.410219 0.582 0.655 0.6940.802 0.8502 0.902 0.9485 0.9451 0.9885 026_695_00300150 Lung 695NCI-H211 456 3.515811 0.644 0.645 0.719 0.8327 0.8177 0.933 0.95190.9812 1.0305 026_721_00302860 Lung 721 NCI-H2029 456 3.591515 0.606 0.80.891 0.9011 0.8579 0.931 0.9429 1.0032 1.0011 026_776_00303250 Lung 776MS-1-L 456 3.614256 0.642 0.767 0.855 0.8458 1.1115 0.929 1.0414 1.02851.162 026_8197_00304741 Lung 8197 LU-139 456 3.667637 0.66 0.84 0.9011.0158 0.9097 0.912 1.0844 1.1048 1.0641 026_702_00302900 Lung 702NCI-H847 456 3.686524 0.818 0.707 0.668 0.6815 0.7184 0.743 0.79990.9145 0.9524 026_8203_00309050 Lung 8203 IST-SL1 456 3.697133 0.6350.801 0.87 0.931 0.9112 1 1.0127 0.9507 1.0704 026_724_00300140 Lung 724NCI-H2081 456 3.857634 0.623 0.866 0.895 0.9027 0.9349 0.948 0.9690.9681 1.0072 026_765_00300050 Lung 765 DMS 273 456 3.890251 0.702 0.8440.904 0.9648 1.028 0.968 1.0795 1.0165 1.0825 026_829_00305160 Lung 829NCI-H2110 456 3.972695 0.67 0.694 0.776 0.8765 0.8765 0.946 1.0573 0.910.9398 026_742_00303230 Lung 742 DMS 53 456 3.990104 0.711 0.802 0.8670.9734 1.0075 1.037 1.0307 1.0204 1.0405 026_710_00316710 Lung 710NCI-H1341 456 4.024081 0.591 0.852 0.7 0.7407 0.8259 0.789 0.8069 0.83860.865 026_738_00302800 Lung 738 NCI-H446 456 4.043153 0.692 0.871 0.9171.0421 0.9341 0.915 1.0657 0.9649 1.0382 026_751_00308570 Lung 751NCI-H209 456 4.061511 0.657 0.848 0.89 0.9403 0.8939 0.91 0.9316 0.95240.9513 026_716_00298900 Lung 716 NCI-H1876 456 4.083964 0.744 0.8660.936 0.9448 0.9881 0.979 0.9782 0.9994 1.0261 026_725_00303280 Lung 725NCI-H2141 456 4.09696 0.711 0.842 0.928 0.9702 0.9155 0.924 1.00441.0101 0.9559 026_688_00302810 Lung 688 SW 1271 456 4.124557 0.602 0.7610.79 0.8093 0.807 0.823 0.896 0.8782 0.9341 026_720_00302380 Lung 720NCI-H1994 456 4.130367 0.696 0.694 0.73 0.8114 0.9245 0.935 0.94840.9393 0.9738 026_811_00311720 Lung 811 NCI-H1435 456 4.138326 0.6350.803 0.855 0.8503 0.8571 0.89 0.8912 0.955 0.9956 026_704_00300250 Lung704 NCI-H1048 456 4.178908 0.166 0.762 0.867 0.8914 0.9318 0.923 0.92011.0122 0.9635 026_746_00302780 Lung 746 SHP-77 456 4.20487 0.79 0.8430.955 1.029 0.9896 1.021 1.0111 0.9931 1.0025 026_829_00311740 Lung 829NCI-H2110 456 4.22315 0.739 0.83 0.914 0.9675 0.9597 0.983 0.9791 1.0320.9995 026_736_00300181 Lung 736 NCI-H69 456 4.26966 0.798 0.87 0.9451.0535 1.0472 1.143 1.0521 1.0846 1.1743 026_724_00303270 Lung 724NCI-H2081 456 4.274134 0.708 0.95 1.023 0.9666 1.0134 0.99 1.0037 1.03881.0531 026_714_00300260 Lung 714 NCI-H1694 456 4.276996 0.701 0.8310.843 0.8708 0.9426 0.93 0.9095 1.1071 1.0848 026_715_00298890 Lung 715NCI-H1836 456 4.291554 0.73 0.833 0.997 0.9315 0.9434 0.955 0.92910.9423 1 026_757_00302870 Lung 757 CPC-N 456 4.345126 0.718 0.819 0.8870.8883 0.9167 0.938 0.9641 1.0154 1.0153 026_8229_00304990 Lung 8229COR-L303 456 4.468573 0.685 0.828 0.788 0.868 0.6881 0.855 0.9579 0.84660.9772 026_691_00308560 Lung 691 NCI-H526 456 4.602758 0.748 0.932 0.9440.9861 0.9744 0.954 1.0035 1.0955 1.0007 026_8099_00316740 Lung 8099LB647-SCLC 456 4.613258 0.755 0.954 0.998 0.9372 0.957 1.041 1.01711.0259 0.9506 026_725_00302750 Lung 725 NCI-H2141 456 4.618451 0.7470.869 0.906 0.9009 0.9283 0.901 1.003 0.9238 1.1424 026_705_00314520Lung 705 NCI-H1092 456 4.678169 0.77 0.861 0.911 1.02 0.9158 1.0820.9997 0.9139 0.961 026_684_00303260 Lung 684 NCI-H1688 456 4.8659470.832 0.831 0.988 1.0672 0.9906 1.099 1.0391 1.0378 1.0698026_725_00300160 Lung 725 NCI-H2141 456 4.894458 0.785 0.908 0.9360.9468 0.9309 0.94 0.9635 1.0477 0.9963 026_8281_00300930 Lung 8281COR-L311 456 5.024667 0.792 0.892 0.947 0.9155 0.8604 0.871 1.02681.0989 1.045 026_757_00300940 Lung 757 CPC-N 456 5.046784 0.796 0.9040.984 1.0041 1.0137 0.919 0.905 0.9063 1.0167 026_701_00309060 Lung 701NCI-H841 456 5.157768 0.826 0.808 0.939 0.891 0.8811 0.919 0.9514 1.04741.0933 026_814_00304750 Lung 814 NCI-H1568 456 5.159278 0.784 0.7260.739 0.745 0.7127 0.774 0.9411 0.9 0.9817 026_741_00305010 Lung 741NCI-H345 456 5.184481 0.801 0.818 0.835 0.9503 0.9116 0.894 0.88490.9853 1.083 026_705_00311700 Lung 705 NCI-H1092 456 5.191642 0.8490.892 0.945 1.0426 0.9184 0.981 1.0366 1.0158 1.0581 026_786_00300950Lung 786 SBC-5 456 5.281883 0.841 1.002 0.991 1.0148 0.9974 0.998 1.02981.0075 1.0015 026_723_00316720 Lung 723 NCI-H2066 456 5.313778 0.7930.815 0.866 0.86 0.9117 0.892 0.9084 0.923 0.9757 026_705_00309080 Lung705 NCI-H1092 456 5.329516 0.829 0.941 0.999 0.9672 0.8963 0.935 0.99910.9888 1.0506 026_709_00305000 Lung 709 NCI-H1304 456 5.330039 0.8380.882 0.906 0.8939 0.9384 0.978 1.0127 1.0322 1.0575 026_739_00314630Lung 739 NCI-H146 456 5.38813 0.858 0.893 0.978 1.0115 1.0583 0.9370.9685 1.0018 1.0831 026_8110_00314450 Lung 8110 LU-165 456 5.5397980.87 0.909 0.986 0.9964 0.9809 1.094 0.9305 0.9513 1.1416026_811_00305140 Lung 811 NCI-H1435 456 5.586661 0.886 0.953 0.9780.9914 1.0102 0.994 0.9871 0.9783 0.9731 026_711_00311710 Lung 711NCI-H1417 456 5.606717 0.875 0.91 0.911 0.9612 1.0374 0.917 1.267 1.01950.9447 026_712_00309090 Lung 712 NCI-H1436 456 5.636542 0.85 0.918 0.9760.9527 0.9331 0.932 0.9583 0.9477 1.0343 026_711_00305130 Lung 711NCI-H1417 456 5.804325 0.849 1.022 0.916 0.9247 0.9771 0.939 0.93261.0168 1.0143 026_728_00311760 Lung 728 NCI-H2196 456 5.845371 0.890.908 0.918 0.9801 0.875 1.033 0.9519 0.931 1.0431 026_831_00311750 Lung831 NCI-H2172 456 5.851811 0.879 0.986 1.002 0.9709 0.9562 0.984 0.97811.0144 0.974 026_1216_00300060 Lung 1216 H292 456 5.909493 0.869 0.8230.808 0.8192 0.8429 0.864 0.9245 0.9789 1.0777 026_8109_00314440 Lung8109 LU-134-A 456 5.930549 0.868 0.95 0.972 0.9375 0.9332 0.959 0.95161.1207 1.1025 026_831_00305170 Lung 831 NCI-H2172 456 6.053261 0.8370.856 0.908 0.8908 0.9397 0.905 0.9677 0.9512 0.9839 026_728_00305180Lung 728 NCI-H2196 456 6.171009 0.9 0.903 0.976 0.9979 0.9199 0.9280.9453 0.9369 0.941 026_689_00300910 Lung 689 NCI-H187 456 6.1915560.887 1.009 0.976 0.9779 0.9535 1.047 1.021 1.0846 0.959026_785_00302770 Lung 785 SBC-1 456 6.234067 0.875 1.037 1.061 0.93761.0385 0.937 0.9403 1.0039 1.1602 026_8280_00306910 Lung 8280 COR-L321456 6.254083 0.965 0.937 1.019 0.8492 0.948 1.048 1.0366 1.0761 0.9586026_706_00318720 Lung 706 NCI-H1105 456 6.355 0.897 0.965 1.009 0.98210.9646 1.037 0.9974 0.9667 0.9589 026_712_00305150 Lung 712 NCI-H1436456 6.498513 0.959 0.818 0.938 1.1033 0.9924 0.922 1.0128 1.0035 0.9263026_8079_00306720 Lung 8079 IST-SL2 456 6.56418 0.947 0.972 0.935 0.96580.9445 0.966 0.9554 1.0191 1.006 026_743_00303220 Lung 743 DMS 114 4566.614771 0.862 0.853 0.785 0.7831 0.861 0.851 0.8815 0.9129 1.0374026_8022_00306711 Lung 8022 COLO-668 456 6.912523 0.97 0.968 0.956 10.9608 0.917 1.0093 0.9217 0.9308 026_8109_00308510 Lung 8109 LU-134-A456 6.98132 0.924 1.012 1.105 0.9946 0.9684 1.081 0.9878 0.9397 0.9606026_758_00303240 Lung 758 HCC-33 456 7.022636 0.931 1.056 1.002 1.08221.0508 1.011 1.0081 1.1612 1.1932 026_771_00306940 Lung 771 Lu-135 4567.040802 0.988 0.969 0.972 1.0271 0.9989 1.006 0.993 1.0649 1.0235026_694_00302790 Lung 694 NCI-H196 456 7.041262 0.976 0.954 0.975 0.95480.9357 0.922 0.9564 0.9433 1.019 026_764_00308550 Lung 764 COR-L95 4567.251052 0.978 1.021 0.944 1.002 1.0708 0.947 0.9637 0.9383 0.903026_763_00300900 Lung 763 COR-L88 456 7.365052 0.96 1.168 0.891 1.14341.1503 0.976 1.0066 1.0636 1.052 026_8134_00306730 Lung 8134 NCI-H64 4567.782872 1.026 1.003 0.976 1.0025 0.951 1.047 0.9338 0.8997 1.0076026_712_00311730 Lung 712 NCI-H1436 456 7.804947 1.019 1.049 0.9470.9403 0.9104 0.931 0.9349 0.9472 0.9659 026_8018_00304801 Lung: NSCLC8018 Calu-6 456 −0.899538 0.106 0.165 0.183 0.2075 0.2699 0.394 0.81820.9121 1.0531 026_1246_00304570 Lung: NSCLC 1246 NCI-H1770 456 −0.0724440.109 0.175 0.267 0.3543 0.4184 0.552 0.6475 1.0183 0.9838026_847_00304580 Lung: NSCLC 847 NCI-H2087 456 1.048994 0.368 0.4020.458 0.4756 0.5065 0.683 0.7888 0.9774 0.9846 026_680_00298830 Lung:NSCLC 680 NCI-H727 456 1.334143 0.329 0.434 0.475 0.4492 0.5064 0.6040.8522 0.9134 0.98 026_748_00304590 Lung: NSCLC 748 NCI-H226 4561.690128 0.342 0.457 0.514 0.5901 0.7687 0.757 0.7854 1.0039 0.932026_851_00298380 Lung: NSCLC 851 CAL-12T 456 1.708943 0.452 0.449 0.490.5839 0.7337 0.842 0.9205 0.938 0.9948 026_861_00300230 Lung: NSCLC 861LCLC-97TM1 456 2.249036 0.424 0.573 0.622 0.7209 0.7517 0.873 1.04120.9724 0.974 026_1245_00304550 Lung: NSCLC 1245 NCI-H1648 456 2.5111310.507 0.571 0.662 0.5623 0.7611 0.961 1.0696 1.0483 1.4204026_1180_00308140 Lung: NSCLC 1180 NCI-H3122 456 2.550953 0.534 0.5170.616 0.6044 0.7162 0.883 0.9512 0.9659 0.9657 026_802_00298451 Lung:NSCLC 802 NCI-H358 456 2.662533 0.509 0.53 0.626 0.7229 0.8072 0.8060.8743 0.9218 0.9534 026_815_00311140 Lung: NSCLC 815 NCI-H1623 4562.802368 0.523 0.592 0.685 0.6168 0.6264 0.746 0.872 0.8493 1.0805026_1180_00302350 Lung: NSCLC 1180 NCI-H3122 456 2.945291 0.724 0.6490.605 0.75 0.7455 0.913 0.9083 1.0538 1.1763 026_8040_00304501 Lung:NSCLC 8040 EKVX 456 3.134434 0.532 0.642 0.782 0.752 0.7074 0.804 0.90871.0521 1.0032 026_865_00308451 Lung: NSCLC 865 COR-L23 456 3.225036 0.580.657 0.735 0.83 0.9489 0.935 1.0144 1.0355 1.0002 026_1243_00304541Lung: NSCLC 1243 NCI-H1395 456 3.23207 0.553 0.739 0.944 0.948 0.92890.926 0.9919 1.0441 1.1113 026_884_00308160 Lung: NSCLC 884 RERF-LC-MS456 3.239905 0.585 0.663 0.662 0.6574 0.7423 0.886 0.9636 0.9621 0.9957026_796_00295871 Lung: NSCLC 796 NCI-H2009 456 3.244878 0.582 0.62 0.6310.635 0.7036 0.747 0.8586 0.9443 0.9943 026_799_00295880 Lung: NSCLC 799NCI-H661 456 3.295179 0.667 0.587 0.685 0.6862 0.8151 0.782 1.00550.9053 0.9863 026_822_00311150 Lung: NSCLC 822 NCI-H1869 456 3.5708330.153 0.716 0.736 0.7399 0.8095 0.819 1.026 0.9674 1.0055026_756_00302670 Lung: NSCLC 756 BEN 456 3.65632 0.639 0.833 0.9420.9322 0.9128 0.965 0.9486 1.0116 1.0192 026_876_00299781 Lung: NSCLC876 LU65 456 3.69068 0.635 0.695 0.704 0.7208 0.8211 0.878 0.9851 1.02111.0551 026_805_00304531 Lung: NSCLC 805 NCI-H1155 456 3.716538 0.610.788 0.787 0.8467 0.9573 0.91 0.953 1.0683 1.0508 026_834_00304610Lung: NSCLC 834 NCI-H2347 456 3.72347 0.653 0.736 0.756 0.8026 0.85240.984 0.9516 1.2251 1.0554 026_822_00304840 Lung: NSCLC 822 NCI-H1869456 3.749478 0.594 0.736 0.791 0.7802 0.8592 0.824 0.8872 0.9703 1.0592026_835_00302390 Lung: NSCLC 835 NCI-H2405 456 3.789193 0.693 0.6590.651 0.6574 0.7337 0.768 0.8849 0.9046 1.0375 026_678_00304981 Lung:NSCLC 678 UMC-11 456 3.815948 0.655 0.751 0.848 0.8851 0.9411 1.0130.9806 1.0842 1.1132 026_807_00314280 Lung: NSCLC 807 NCI-H650 4563.884087 0.604 0.778 0.772 0.8577 0.8346 0.815 1.0068 0.831 0.9772026_871_00299771 Lung: NSCLC 871 LK-2 456 3.898613 0.684 0.806 0.9030.9071 0.958 1.018 0.9981 1.0604 1.0786 026_1249_00308150 Lung: NSCLC1249 NCI-H720 456 3.907633 0.632 0.829 0.932 1.0847 1.526 0.799 0.87511.2042 1.1215 026_8231_00304510 Lung: NSCLC 8231 EMC-BAC-1 456 3.9439460.675 0.713 0.751 0.8781 0.831 0.931 0.976 1.0569 1.0459026_820_00304560 Lung: NSCLC 820 NCI-H1755 456 3.964037 0.658 0.7690.785 0.8744 0.8799 0.931 0.9563 1.1261 1.0206 026_815_00304830 Lung:NSCLC 815 NCI-H1623 456 3.981628 0.646 0.709 0.712 0.7108 0.8124 0.8790.7844 1.0245 1.0786 026_839_00302410 Lung: NSCLC 839 SW 900 4563.982367 0.65 0.725 0.782 0.8242 0.8576 0.897 0.9535 1.0086 0.9978026_804_00308480 Lung: NSCLC 804 NCI-H810 456 4.03318 0.731 0.763 0.910.9442 0.9087 1.003 0.9482 0.9716 1.0139 026_678_00309011 Lung: NSCLC678 UMC-11 456 4.0744 0.514 0.846 0.86 0.931 0.9037 0.925 0.9312 0.93541.0786 026_842_00298540 Lung: NSCLC 842 NCI-H520 456 4.082969 0.7290.811 0.916 1.0516 1.0142 1.002 1.0457 1.0025 0.9761 026_824_00314260Lung: NSCLC 824 NCI-H1944 456 4.107799 0.695 0.731 0.691 0.7704 0.8930.886 0.905 0.9791 1.0866 026_888_00298370 Lung: NSCLC 888 ABC-1 4564.123019 0.64 0.676 0.717 0.7343 0.8328 0.84 0.8774 0.9195 0.9448026_823_00298430 Lung: NSCLC 823 NCI-H1915 456 4.260241 0.691 0.7070.702 0.7393 0.7957 0.834 0.9053 0.9612 0.9902 026_8232_00304520 Lung:NSCLC 8232 EMC-BAC-2 456 4.327709 0.712 0.795 0.825 0.8558 0.8815 0.9590.9014 1.1842 1.0135 026_755_00300611 Lung: NSCLC 755 NCI-H1975 4564.450033 0.666 0.968 0.963 0.9052 0.9346 0.928 0.9969 0.9681 0.973026_868_00295440 Lung: NSCLC 868 PC-14 456 4.458594 0.726 0.893 0.8420.8971 0.9361 1.057 0.9146 1.1281 0.933 026_872_00299750 Lung: NSCLC 872HARA 456 4.480998 0.731 0.742 0.798 0.8152 0.8972 0.941 0.9735 1.00741.0301 026_800_00298441 Lung: NSCLC 800 NCI-H23 456 4.511394 0.702 0.6980.786 0.7938 0.8549 0.889 0.9216 0.9447 1.013 026_836_00304620 Lung:NSCLC 836 NCI-H2444 456 4.530511 0.684 0.798 0.819 0.8507 0.863 0.860.9116 1.0106 1.0518 026_865_00296401 Lung: NSCLC 865 COR-L23 4564.577187 0.791 0.78 0.815 0.8605 0.9148 0.924 1.1179 1.1287 1.1293026_858_00300591 Lung: NSCLC 858 HCC-78 456 4.583505 0.703 0.983 0.9361.06 0.9813 0.96 1.0028 0.9854 1.0584 026_854_00300681 Lung: NSCLC 854EPLC-272H 456 4.590654 0.736 0.914 0.943 1.0019 1.0017 0.864 0.89370.9686 1.0373 026_837_00311160 Lung: NSCLC 837 NCI-H2122 456 4.5951920.771 0.917 0.969 0.9878 0.9667 1.017 1.0165 1.0062 1.0324026_8111_00308110 Lung: NSCLC 8111 LXF-289 456 4.689278 0.711 0.7960.884 0.8648 0.868 0.89 0.9108 0.8979 0.9642 026_1136_00308471 Lung:NSCLC 1136 NCI-H1993 456 4.705234 0.725 0.768 0.682 0.7469 0.7866 0.8830.9296 0.914 0.9652 026_827_00308860 Lung: NSCLC 827 NCI-H2085 4564.711775 0.79 0.823 0.87 0.9311 0.924 1.007 0.967 1.0124 1.1093026_859_00311090 Lung: NSCLC 859 HCC-827 456 4.735253 0.737 0.86 0.8520.7863 0.8298 0.853 1.0366 1.1949 0.9783 026_886_00296261 Lung: NSCLC886 EBC-1 456 4.744821 0.761 0.848 0.884 0.9053 0.9307 0.969 0.99030.9432 1.0947 026_8132_00308130 Lung: NSCLC 8132 NCI-H2126 456 4.7816350.749 0.796 0.839 0.8331 0.8916 0.939 0.9049 0.9661 0.9286026_793_00299711 Lung: NSCLC 793 NCI-H1781 456 4.904802 0.775 0.8010.686 0.7429 0.7804 0.897 0.9136 0.9608 1.0112 026_1247_00314271 Lung:NSCLC 1247 NCI-H2291 456 4.945975 0.773 0.893 0.836 0.831 0.851 1.0291.0615 0.9828 1.0493 026_860_00298400 Lung: NSCLC 860 LCLC-103H 4564.97209 0.751 0.819 0.803 0.8985 0.8725 0.898 0.9606 0.9519 1.061026_806_00300270 Lung: NSCLC 806 NCI-H647 456 5.044917 0.792 0.845 0.8820.8698 0.8856 0.881 1.0769 0.9333 1.0648 026_877_00300630 Lung: NSCLC877 PC-3 [JPC-3] 456 5.056812 0.847 0.832 0.968 0.9943 1.0985 1.011.0152 1.0063 1.1608 026_753_00298460 Lung: NSCLC 753 NCI-H460 4565.061076 0.839 0.888 0.945 0.9015 1.041 1.039 1.021 0.9905 1.0126026_844_00295461 Lung: NSCLC 844 SW 1573 456 5.077017 0.798 0.834 0.8330.8824 0.8946 0.971 1.0223 0.9943 1.0074 026_8088_00314320 Lung: NSCLC8088 KNS-62 456 5.130485 0.86 0.847 0.879 0.9874 0.9738 1.047 1.06021.0303 0.9876 026_848_00300641 Lung: NSCLC 848 SK-LU-1 456 5.2771 0.8110.91 0.946 0.9174 0.9429 0.922 0.9544 1.0372 1.0115 026_864_00304961Lung: NSCLC 864 COR-L 105 456 5.319498 0.718 0.78 0.762 0.8018 0.76830.837 0.9721 0.8644 0.9527 026_677_00298361 Lung: NSCLC 677 A549 4565.414187 0.763 0.799 0.843 0.8595 0.9001 0.919 0.9405 0.9783 0.996026_8207_00304810 Lung: NSCLC 8207 LC-1F 456 5.423228 0.825 0.794 0.8450.8457 0.8483 0.976 0.8421 0.8626 0.9602 026_833_00299790 Lung: NSCLC833 NCI-H2342 456 5.548934 0.856 0.888 0.917 0.9241 0.9293 1.015 0.94491.0616 0.9624 026_870_00299801 Lung: NSCLC 870 RERF-LC-KJ 456 5.6855110.892 0.96 0.999 0.9623 1.0469 1.054 1.0305 1.0128 1.0476026_845_00311341 Lung: NSCLC 845 NCI-H1838 456 5.711103 0.847 0.8670.909 0.9088 0.9208 0.932 0.9775 0.9857 0.9755 026_8103_00306210 Lung:NSCLC 8103 LC-2-ad 456 5.718708 0.836 0.96 1.004 0.9427 1.0257 1.0641.0682 0.9834 1.007 026_794_00300601 Lung: NSCLC 794 NCI-H1792 4565.721238 0.339 0.904 0.992 0.9248 0.9615 1.005 0.9405 0.9595 1.0225026_890_00300220 Lung: NSCLC 890 H3255 456 5.722728 0.903 0.862 0.8440.9926 1.005 0.998 1.1503 1.1306 1.1501 026_862_00304820 Lung: NSCLC 862LOU-NH91 456 5.738178 0.886 0.864 0.837 0.9386 0.9258 0.887 0.948 1.05611.1752 026_812_00298410 Lung: NSCLC 812 NCI-H1437 456 5.791603 0.8940.946 0.993 0.9231 0.9704 1.003 1.0591 1.0305 1.0361 026_862_00316560Lung: NSCLC 862 LOU-NH91 456 5.867157 0.871 0.905 0.923 0.9415 0.91430.955 0.9491 0.9721 0.9923 026_857_00296270 Lung: NSCLC 857 HCC-44 4565.930045 0.904 0.868 0.893 0.9249 0.9348 0.956 1.0259 0.9727 1.0027026_679_00296240 Lung: NSCLC 679 ChaGo-K-1 456 5.965866 0.878 0.9150.949 0.8968 0.942 0.953 0.9732 0.9711 1.0338 026_791_00298421 Lung:NSCLC 791 NCI-H1650 456 5.971987 0.888 0.896 0.979 0.9429 0.9543 0.9210.9516 0.9551 0.9966 026_864_00311551 Lung: NSCLC 864 COR-L 105 4565.975297 0.765 0.838 0.843 0.8384 0.8225 0.848 0.8917 0.8749 0.9811026_846_00300620 Lung: NSCLC 846 NCI-H2030 456 6.014339 0.886 0.9790.985 0.9316 1.0428 0.925 0.9778 1.056 0.9904 026_841_00296291 Lung:NSCLC 841 NCI-H2170 456 6.01763 0.897 0.938 0.928 0.9356 0.9557 0.9551.0968 1.0708 1.0963 026_813_00308231 Lung: NSCLC 813 NCI-H1563 4566.06364 0.856 0.979 0.977 0.9369 0.9242 0.912 0.8859 0.9059 0.8856026_816_00295860 Lung: NSCLC 816 NCI-H1651 456 6.160326 0.864 1.0391.026 1.008 1.0776 0.871 1.0553 1.0199 0.9978 026_818_00308241 Lung:NSCLC 818 NCI-H1703 456 6.172519 0.946 0.829 0.858 0.9042 0.9197 0.8870.9097 0.9729 0.9634 026_808_00296300 Lung: NSCLC 808 NCI-H838 4566.172929 0.922 0.941 0.955 1.022 0.964 1.043 1.0358 1.0134 1.0658026_855_00302330 Lung: NSCLC 855 HCC-15 456 6.195295 0.868 0.911 0.9010.8895 0.9071 0.95 0.9608 1.0091 1.0383 026_803_00298470 Lung: NSCLC 803NCI-H522 456 6.200929 0.878 0.933 0.953 0.8695 0.9015 0.833 0.88820.8789 1.1066 026_832_00303110 Lung: NSCLC 832 NCI-H2228 456 6.2405860.908 0.872 0.907 0.9005 0.9724 0.974 0.9776 0.9706 0.968026_874_00308171 Lung: NSCLC 874 RERF-LC-Sq1 456 6.282697 1.064 0.8540.829 0.8009 0.8914 0.943 0.8612 0.9931 0.9393 026_879_00308101 Lung:NSCLC 879 LU99A 456 6.327465 0.881 0.987 0.995 1.0199 0.9725 0.9480.9588 0.9756 0.9692 026_856_00299760 Lung: NSCLC 856 HCC-366 4566.328052 0.936 0.943 0.959 1.0286 0.9991 1.002 1.0072 1.0077 1.0015026_798_00304790 Lung: NSCLC 798 Calu-3 456 6.338698 0.926 0.986 0.8690.9635 1.0683 1.166 1.1053 1.1123 1.1003 026_825_00308250 Lung: NSCLC825 NCI-H2023 456 6.435057 0.938 0.885 0.929 0.961 0.9166 0.959 0.95020.9835 0.9846 026_8072_00306200 Lung: NSCLC 8072 HOP-62 456 6.4817740.973 0.958 0.931 0.9405 0.9799 0.901 1.0374 0.9717 1.1512026_843_00296331 Lung: NSCLC 843 SK-MES-1 456 6.499026 0.91 0.99 0.9340.9509 0.9694 1.003 0.9804 0.9674 1.0295 026_1251_00318710 Lung: NSCLC1251 NCI-H835 456 6.684349 0.964 1.054 0.854 0.9853 0.77 0.963 0.87140.9637 1.1416 026_816_00302370 Lung: NSCLC 816 NCI-H1651 456 6.7694330.903 1.072 1.021 1.002 1.0583 0.94 1.0664 1.0462 1.0774026_801_00295941 Lung: NSCLC 801 NCI-H1299 456 6.808075 1.036 0.8980.912 0.9194 0.9832 0.955 1.0252 0.9946 1.085 026_850_00311050 Lung:NSCLC 850 201T 456 6.874432 0.942 0.957 0.988 0.989 0.9596 0.985 0.97590.9839 0.9726 026_818_00309001 Lung: NSCLC 818 NCI-H1703 456 7.1924090.889 0.857 0.855 0.8355 0.8207 0.856 0.8191 0.9124 0.9468026_1247_00304851 Lung: NSCLC 1247 NCI-H2291 456 7.252153 1.162 1.0470.877 1.034 0.8759 0.6949 1.2149 1.0391 1.1021 026_819_00306250 Lung:NSCLC 819 NCI-H1734 456 7.342705 1.046 1.007 1.012 1.018 0.9902 0.9450.9742 0.9718 1.0221 026_821_00306261 Lung: NSCLC 821 NCI-H1793 4567.345651 1.015 1.1 1.058 1.0171 1.0121 1.065 10.342 1.0776 1.0468026_798_00308080 Lung: NSCLC 798 Calu-3 456 7.389624 1.032 1 0.9140.9927 1.0342 1.004 1.0095 0.9327 0.938 026_678_00306181 Lung: NSCLC 678UMC-11 456 7.458364 1.03 1.058 1.054 1.0958 1.0021 1.03 1.0784 1.07791.0688 026_797_00308730 Lung: NSCLC 797 NCI-H596 456 7.556662 1.1241.076 0.92 0.9992 0.9285 0.936 0.911 1.0276 0.8862 026_790_00306231Lung: NSCLC 790 NCI-H1573 456 7.606756 1.049 1 1.054 1.0146 1.0291 0.991.0304 1.0221 1.0512 026_752_00306191 Lung: NSCLC 752 A-427 456 7.6084441.109 0.988 1.054 1.0224 0.9429 1.084 1.0956 1.0273 1.0528026_845_00306271 Lung: NSCLC 845 NCI-H1838 456 7.666847 1.074 1.08 1.0641.0779 1.0805 1.046 1.0674 0.9941 1.02 026_8133_00306280 Lung: NSCLC8133 NCI-H322M 456 7.713112 1.158 1.06 1.135 1.1499 1.1305 1.103 1.14641.1475 1.0913 026_8130_00306220 Lung: NSCLC 8130 NCI-H1355 456 7.7658881.12 1.188 1.002 0.9001 0.8103 1.095 0.9608 0.9714 0.9997026_683_00306241 Lung: NSCLC 683 NCI-H1581 456 7.774034 1.163 1.0240.949 1.0959 1.0605 1.114 1.0118 0.9537 0.9212 026_792_00306140 Lung:NSCLC 792 NCI-H1666 456 7.825548 1.136 1.069 1.132 1.0901 1.0688 1.0971.0684 1.0463 1.1115 026_840_00306151 Lung: NSCLC 840 NCI-H441 4567.946249 1.088 1.059 1.052 1.0622 1.0476 1.049 1.0439 1.045 1.0714026_8075_00306131 Lung: NSCLC 8075 IA-LM 456 7.984543 1.046 1.042 1.0431.0424 1.0323 1.048 1.0441 1.0279 1.0694 026_61_00285570 Lymphoma 61JSC-1 456 0.316646 0.313 0.329 0.341 0.3908 0.5654 0.63 0.724 0.83460.8527 026_8222_00291350 Lymphoma 8222 H9 456 0.402947 0.102 0.284 0.2720.3996 0.5762 0.732 0.8412 0.8346 0.8529 026_140_00291320 Lymphoma 140A3/KAW 456 0.717935 0.185 0.296 0.408 0.4762 0.5561 0.736 0.8297 0.87610.8425 026_237_00291380 Lymphoma 237 SU-DHL-16 456 0.982467 0.155 0.2780.405 0.5737 0.6759 0.816 0.9129 0.9363 0.9318 026_220_00288720 Lymphoma220 OCI-LY-19 456 1.28484 0.324 0.393 0.455 0.5338 0.6443 0.826 0.93430.9969 1.073 026_257_00285640 Lymphoma 257 WIL2 NS 456 1.523653 0.320.456 0.509 0.6199 0.6926 0.771 0.8518 0.9524 0.934 026_239_00288750Lymphoma 239 SU-DHL-5 456 1.616577 0.174 0.577 0.461 0.7856 0.7368 0.8450.9512 0.9535 1.2005 026_124_00287850 Lymphoma 124 BC-1 456 1.8167660.239 0.53 0.6 0.6778 0.8612 0.946 0.9754 0.9846 1.0038 026_104_00287960Lymphoma 104 TUR 456 2.217781 0.396 0.51 0.597 0.7468 0.9062 0.94 0.98220.9761 1.0165 026_8199_00291330 Lymphoma 8199 CTB-1 456 2.341488 0.4450.509 0.654 0.6993 0.7792 0.767 0.8214 0.8543 0.9676 026_69_00283480Lymphoma 69 CA46 456 2.43077 0.358 0.6 0.775 0.78 0.8817 0.896 0.99120.9446 0.9358 026_112_00285620 Lymphoma 112 SR 456 2.466522 0.224 0.5730.653 0.7668 0.8482 0.866 0.9087 0.9409 0.9255 026_241_00288760 Lymphoma241 SU-DHL-8 456 2.509783 0.398 0.583 0.743 0.8249 0.935 0.956 1.04390.9903 1.0527 026_255_00285610 Lymphoma 255 Sci-1 456 2.705483 0.4450.61 0.788 0.9473 0.9153 1.003 0.9035 0.8642 0.8757 026_62_00293930Lymphoma 62 IM-9 456 2.809638 0.572 0.562 0.594 0.6839 0.7773 1.18 0.8761.0089 1.0329 026_93_00287880 Lymphoma 93 HH 456 2.828886 0.523 0.5750.731 0.804 0.9283 0.948 0.9965 0.999 1.0069 026_216_00290710 Lymphoma216 NU-DUL-1 456 2.880782 0.459 0.664 0.767 0.8093 0.7784 0.884 0.93380.9363 0.9519 026_123_00287860 Lymphoma 123 BC-3 456 2.893681 0.4950.678 0.776 0.9051 1.0108 1.003 1.0056 0.9841 0.9941 026_113_00288520Lymphoma 113 DB 456 2.901204 0.479 0.705 0.868 0.9097 0.9559 0.9841.0104 0.992 1.0316 026_8035_00303290 Lymphoma 8035 DOHH-2 456 2.9274610.508 0.78 0.831 0.8009 0.9998 0.997 1.0295 0.9393 1.0203026_240_00300290 Lymphoma 240 SU-DHL-6 456 2.933827 0.208 0.663 0.7330.7473 0.7814 0.839 0.8554 0.9327 1.029 026_248_00285630 Lymphoma 248VAL 456 2.964017 0.528 0.639 0.739 0.9112 0.9265 0.96 1.0023 0.98270.9859 026_162_00290790 Lymphoma 162 HDLM-2 456 2.980685 0.56 0.8110.678 0.7341 0.7627 0.782 0.7915 0.9638 0.7387 026_105_00287940 Lymphoma105 RPMI 6666 456 3.064381 0.516 0.701 0.691 0.7257 0.8234 0.904 0.98831.0013 1.033 026_240_00302920 Lymphoma 240 SU-DHL-6 456 3.114948 0.3260.629 0.741 0.7802 0.8119 0.831 0.9347 0.921 0.9838 026_163_00287730Lymphoma 163 HD-MY-Z 456 3.123344 0.582 0.606 0.654 0.6825 0.785 0.8960.9395 0.98 1.0207 026_139_00283530 Lymphoma 139 MC116 456 3.12577 0.0850.686 0.917 0.9565 0.9611 0.99 0.9875 0.9789 0.9559 026_133_00293941Lymphoma 133 NK-92MI 456 3.297366 0.637 0.637 0.757 0.8911 0.8633 1.1361.0548 0.89 0.9001 026_282_00287750 Lymphoma 282 P32/ISH 456 3.3282030.416 0.744 0.875 0.8947 0.8754 0.907 0.9959 0.9144 0.9277026_80_00287950 Lymphoma 80 ST486 456 3.520498 0.263 0.688 0.699 0.79690.8071 0.904 0.8902 0.9142 1.017 026_73_00283500 Lymphoma 73 EB-3 4563.528971 0.566 0.739 0.806 0.8214 0.8547 1.09 0.8321 0.8619 0.8548026_60_00287891 Lymphoma 60 JM1 456 3.618767 0.577 0.678 0.734 0.79320.8209 0.833 0.8444 0.8567 0.902 026_185_00288820 Lymphoma 185 L-540 4563.666174 0.626 0.88 0.936 1.0302 1.0245 1.037 1.0524 1.0175 1.0525026_70_00283490 Lymphoma 70 Daudi 456 3.687192 0.603 0.789 0.925 0.88670.9929 0.987 0.8586 0.9555 0.8243 026_228_00291370 Lymphoma 228 RC-K8456 3.710403 0.605 0.678 0.732 0.7776 0.8387 0.943 0.9437 0.9368 0.9149026_74_00285600 Lymphoma 74 Raji 456 3.736373 0.639 0.768 0.89 0.82580.9671 0.97 0.9444 0.9665 1.046 026_173_00288920 Lymphoma 173 KARPAS-422456 3.812903 0.638 0.787 0.876 0.8937 0.8652 0.959 0.9976 0.9896 1.005026_125_00287910 Lymphoma 125 MC/CAR 456 3.860302 0.687 0.792 0.8730.8871 1.009 1.011 0.9976 0.987 1.0162 026_280_00285420 Lymphoma 280SCC-3 456 3.903427 0.648 0.86 0.895 0.9301 0.9335 0.934 1.0707 0.90.9445 026_242_00288770 Lymphoma 242 SUP-HD1 456 4.023177 0.671 0.8150.875 0.8573 0.8914 0.965 1.0137 0.9754 1.1043 026_160_00285410 Lymphoma160 GRANTA-519 456 4.112789 0.631 0.706 0.761 0.7798 0.7991 0.771 0.91670.9366 0.9971 026_144_00287710 Lymphoma 144 BL-41 456 4.441898 0.5361.076 0.941 0.9045 0.8922 0.893 0.9542 0.9696 1.0289 026_184_00290800Lymphoma 184 L-428 456 4.489131 0.614 0.705 0.723 0.7315 0.7351 0.7211.0011 0.7501 0.8687 026_128_00290690 Lymphoma 128 Farage 456 4.5851560.766 0.834 0.861 0.8151 0.9324 0.908 1.3723 1.1697 1.2229026_250_00285660 Lymphoma 250 WSU-NHL 456 4.693667 0.719 0.765 0.8660.8242 0.9178 0.947 0.9725 0.9585 0.9714 026_182_00290700 Lymphoma 182L-1236 456 4.714251 0.76 0.859 0.875 0.8522 0.9702 0.962 1.0483 0.94321.3018 026_95_00283510 Lymphoma 95 HT 456 4.721481 0.719 0.842 0.880.8266 0.8534 0.902 1.0348 0.9564 0.966 026_264_00288840 Lymphoma 264 TK456 4.750226 0.794 0.966 0.936 0.9709 1.0212 1.024 1.0348 1.0318 1.0681026_266_00287760 Lymphoma 266 SLVL 456 4.90866 0.731 0.786 0.794 0.80120.8742 0.873 0.9435 0.9199 0.977 026_75_00282830 Lymphoma 75 Jiyoye 4564.937844 0.801 0.99 0.974 1.0523 1.196 1.107 1.0641 1.1044 1.0749026_111_00291360 Lymphoma 111 Hs 445 456 5.022248 0.704 0.734 0.7030.794 0.7451 0.779 0.8243 0.8675 0.946 026_8151_00288540 Lymphoma 8151Ramos-2G6-4C10 456 5.147746 0.242 0.868 0.954 0.9827 1.022 0.974 0.99670.9674 1.0529 026_156_00287720 Lymphoma 156 DG-75 456 5.222394 0.8341.001 1.03 1.0349 0.9848 0.996 1.012 0.9965 0.9857 026_243_00296620Lymphoma 243 SUP-M2 456 5.25553 0.648 0.88 0.96 0.9351 0.9518 0.9680.9742 0.9627 0.9659 026_86_00287870 Lymphoma 86 EB2 456 5.287284 0.861.017 1.01 1.0356 1.0217 1.05 1.0267 1.0118 1.0227 026_162_00288790Lymphoma 162 HDLM-2 456 5.425219 0.877 0.903 0.797 0.9228 1.0293 1.0431.0157 1.0517 1.0837 026_172_00287740 Lymphoma 172 KARPAS-299 4565.433867 0.832 0.891 0.95 0.9494 0.955 0.954 0.9499 0.9633 0.9487026_235_00285430 Lymphoma 235 SU-DHL-1 456 6.401484 0.885 0.997 0.9870.9424 0.9387 0.884 0.8642 0.9464 0.9439 026_143_00288690 Lymphoma 143AMO-1 456 6.692908 0.924 1.025 1.129 1.034 1.1112 1.034 1.0278 1.01361.0255 026_193_00288830 Lymphoma 193 MHH-PREB-1 456 6.799634 0.622 0.9581.012 1.0149 1.0368 1.025 1.0064 0.9803 0.989 026_236_00288730 Lymphoma236 SU-DHL-10 456 6.99662 0.175 0.993 1.05 1.0763 1.0476 1.029 1.05091.0397 1.0494 026_81_00288530 Lymphoma 81 GA-10 456 7.031749 0.608 1.0031.009 1.1031 1.0252 1.021 1.0113 1.0077 1.0848 026_251_00287840 Lymphoma251 YT 456 7.411746 0.64 0.986 1.008 1.0201 1.0204 1.022 1.0267 0.98481.0079 026_178_00288810 Lymphoma 178 KM-H2 456 7.739504 1.008 1.0090.976 1.0197 1.0404 1.036 1.0412 1.0156 1.0276 026_249_00285650 Lymphoma249 WSU-DLCL2 456 8.212885 1.047 1.226 1.172 1.1142 1.0869 1.071 0.98371.0247 1.0207 026_238_00288740 Lymphoma 238 SU-DHL-4 456 8.647656 1.0611.077 1.045 1.0257 1.0384 1.029 1.0236 1.018 1.0204 026_131_00287930Lymphoma 131 RL 456 8.679308 1.124 1.094 1.048 1.024 1.0293 1.012 1.02930.9824 1.0061 026_915_00269070 Miscellaneous 915 Hs 633T 456 1.7979430.344 0.532 0.607 0.6061 0.6357 0.736 0.8936 1.0346 1.1375026_911_00269060 Miscellaneous 911 GCT 456 2.424125 0.433 0.617 0.6420.6508 0.655 0.804 0.7763 0.9066 0.9967 026_8172_00269460 Miscellaneous8172 SW872 456 3.196748 0.511 0.716 0.719 0.7459 0.8264 0.881 0.91070.9313 1.0224 026_913_00271970 Miscellaneous 913 JAR 456 3.876744 0.6850.695 0.794 0.851 0.9936 1.011 0.9013 1.0881 1.0491 026_8194_00271980Miscellaneous 8194 JEG-3 456 3.901223 0.623 0.808 0.876 0.9097 0.90370.874 0.9014 0.943 1.1354 026_8171_00269120 Miscellaneous 8171 SW684 4563.978538 0.693 0.91 0.98 1.1114 1.0275 1.032 0.9881 0.9218 1.0725026_8112_00269450 Miscellaneous 8112 MFH-ino 456 4.013788 0.691 0.6740.795 0.8068 0.8378 0.91 0.9824 1.0389 1.0625 026_916_00271320Miscellaneous 916 HT 1080 456 4.254215 0.695 0.804 0.766 0.7943 0.82760.873 1.1135 1.1006 1.1093 026_8004_00271280 Miscellaneous 8004 A388 4564.494172 0.743 0.793 0.841 0.8441 0.8899 0.888 1.1408 0.925 1.1327026_8112_00271340 Miscellaneous 8112 MFH-ino 456 4.624376 0.721 0.7320.741 0.7501 0.7753 0.799 1.017 0.9044 1.0866 026_8004_00269400Miscellaneous 8004 A388 456 4.778414 0.747 0.764 0.776 0.8164 0.86280.861 0.9217 0.8924 1.0697 026_8192_00269470 Miscellaneous 8192 VA-ES-BJ456 4.956405 0.78 0.867 0.874 0.984 0.9192 0.889 0.9737 1.0227 1.0186026_8194_00269440 Miscellaneous 8194 JEG-3 456 5.058221 0.786 0.87 1.0480.908 0.8876 0.984 0.8804 1.0294 1.0609 026_913_00269430 Miscellaneous913 JAR 456 5.170211 0.816 0.841 0.849 0.8709 0.8661 0.88 1.0502 1.05851.0591 026_916_00269420 Miscellaneous 916 HT 1080 456 5.302771 0.8310.891 0.904 0.9053 0.9327 0.978 0.9465 1.1023 1.1108 026_8175_00269140Miscellaneous 8175 SW982 456 5.586364 0.832 0.968 0.988 1.1946 1.06381.017 1.0728 1.1006 1.2781 026_1225_00269660 Muscle 1225 RD 456 2.6942070.612 0.595 0.602 0.575 0.6051 0.678 0.8404 0.9183 0.947 26_135_00271680Muscle 135 SJCRH30 456 2.740947 0.247 0.61 0.713 0.8654 0.8633 0.8540.9433 0.9991 1.105 026_924_00269640 Muscle 924 A673 456 2.779569 0.5630.554 0.607 0.6884 0.7085 0.808 0.8122 0.8796 0.9437 26_562_00271660Muscle 562 KYM-1 456 2.818237 0.433 0.665 0.727 0.7663 0.7943 0.8350.9132 0.9948 0.9596 026_135_00271410 Muscle 135 SJCRH30 456 3.0593850.345 0.672 0.79 0.8395 0.8741 0.957 0.9768 1.032 1.1796026_923_00269680 Muscle 923 RH-41 456 3.133389 0.309 0.65 0.731 0.78950.6971 0.795 0.7303 0.9458 0.9325 026_562_00270060 Muscle 562 KYM-1 4563.396905 0.586 0.748 0.78 0.8707 0.8698 0.955 1.0372 1.0323 1.0217026_920_00269670 Muscle 920 RH-1 456 3.433085 0.581 0.772 0.846 0.90320.9238 0.951 0.9496 0.9615 0.9852 026_135_00270070 Muscle 135 SJCRH30456 3.759361 0.326 0.727 0.825 0.8737 0.9084 0.931 0.9701 0.9946 0.9435026_919_00269630 Muscle 919 A-204 456 5.25605 0.778 0.798 0.868 0.87440.861 0.89 0.9735 0.9392 0.9765 026_921_00285151 Muscle 921 RH-18 4565.863422 0.84 0.987 0.961 0.9653 0.9811 0.903 0.8875 0.9228 0.9815026_8182_00293791 Muscle 8182 TE-441-T 456 6.133177 0.848 1.001 1.1031.0954 1.1007 0.855 0.8882 1.0698 0.8069 026_369_00258500 Nervous System369 CHP-212 456 −4.151196 0.165 0.184 0.183 0.1873 0.1985 0.219 0.29480.3285 0.4946 026_390_00262920 Nervous System 390 NB69 456 −1.3827 0.0560.074 0.068 0.0948 0.096 0.163 0.4343 0.6938 0.8833 026_629_00316570Nervous System 629 NB(TU)1-10 456 −0.654294 0.322 0.361 0.388 0.3980.4347 0.594 0.7533 0.6321 0.6782 026_366_00257090 Nervous System 366BE(2)-M17 456 −0.258429 0.204 0.352 0.333 0.3872 0.4128 0.547 0.64150.7687 0.9298 026_384_00314240 Nervous System 384 MHH-NB-11 456 1.3161820.368 0.42 0.48 0.6318 0.5903 0.783 0.7802 0.9243 0.5317026_385_00314290 Nervous System 385 SIMA 456 1.359572 0.402 0.496 0.4880.5135 0.6006 0.629 0.8787 0.8808 1.0864 026_630_00264810 Nervous System630 NH-12 456 1.516245 0.143 0.443 0.537 0.5763 0.6816 0.684 0.72630.9504 0.791 026_8124_00308720 Nervous System 8124 NB14 456 1.7843370.802 0.401 0.6 0.6379 0.6871 0.943 0.8827 0.8247 0.9343026_639_00271120 Nervous System 639 GOTO 456 1.784838 0.174 0.52 0.540.6353 0.6485 0.726 0.9341 0.8908 1.1295 026_8094_00314230 NervousSystem 8094 LAN-6 456 1.824343 0.55 0.565 0.476 0.5734 0.6862 0.6560.7893 0.9264 0.7016 026_8076_00260401 Nervous System 8076 IMR-5 4561.864038 0.346 0.489 0.575 1.2246 0.8619 0.823 1.0781 0.7607 0.8894026_8127_00258950 Nervous System 8127 NB5 456 2.106125 0.202 0.628 0.6110.6823 0.725 0.845 0.9541 0.9703 1.0012 026_8124_00311130 Nervous System8124 NB14 456 2.216808 0.544 0.563 0.516 0.5711 0.5857 0.776 0.90140.9091 1.0395 026_8126_00264800 Nervous System 8126 NB17 456 2.2895460.347 0.54 0.684 0.7567 0.8851 0.929 0.8729 0.9752 0.9196026_641_00308180 Nervous System 641 TGW 456 2.326645 0.456 0.502 0.5930.7234 0.8006 0.88 0.9296 0.9651 0.9821 026_8121_00256290 Nervous System8121 NB10 456 2.356578 0.646 0.568 0.562 0.5835 0.6642 0.613 0.54870.6035 1.0005 026_8220_00308810 Nervous System 8220 KP-N-YN 456 2.4704650.366 0.619 0.752 0.7815 0.7642 0.87 0.8457 0.8335 0.8942026_8090_00263920 Nervous System 8090 KP-N-YS 456 2.547617 0.267 0.5830.69 0.9135 0.905 0.924 0.9505 0.9232 1.012 026_363_00252730 NervousSystem 363 SK-N-FI 456 2.667265 0.519 0.583 0.574 0.6332 0.5059 0.5710.7766 0.6898 0.9188 026_382_00311110 Nervous System 382 KELLY 4563.39659 0.518 0.757 0.767 0.7123 0.7538 0.883 0.8718 0.8733 0.9755026_8064_00259120 Nervous System 8064 GI-ME-N 456 3.426922 0.582 0.7630.78 0.8295 0.8371 0.955 1.0539 1.0652 1.104 026_8195_00318640 NervousSystem 8195 CHP-134 456 3.519288 0.6 0.8 0.86 0.909 0.9931 0.919 0.95330.941 1.0273 026_382_00308690 Nervous System 382 KELLY 456 3.678148 0.590.706 0.666 0.7413 0.8277 0.814 0.8335 0.8742 0.9843 026_8129_00314250Nervous System 8129 NB7 456 3.824042 0.644 0.861 0.945 0.972 0.96550.998 1.0941 1.0771 1.0471 026_8126_00280241 Nervous System 8126 NB17456 3.845261 0.611 0.771 0.746 1.0569 0.8567 0.864 0.9408 0.9041 0.972026_8007_00252850 Nervous System 8007 ACN 456 3.989797 0.411 0.72 0.8210.7359 0.7717 0.797 0.8528 1.0702 0.9564 026_8122_00273460 NervousSystem 8122 NB12 456 4.358809 0.15 0.809 0.911 0.9912 0.9113 1.0210.9055 0.8723 1.0083 026_8226_00252530 Nervous System 8226 NBsusSR 4564.365592 1.14 0.81 0.878 0.6567 0.6737 0.94 0.7615 0.8399 1.1136026_8064_00256670 Nervous System 8064 GI-ME-N 456 4.809816 0.398 0.8710.839 0.9402 0.8757 0.96 0.9284 0.9838 1.0451 026_8128_00257200 NervousSystem 8128 NB6 456 4.886145 0.836 0.93 0.956 1.0121 0.9926 1.008 0.98930.9944 0.9472 026_8123_00256300 Nervous System 8123 NB13 456 5.7752540.837 1.028 1.034 0.9756 1.0272 0.974 0.9893 0.9781 0.9486026_396_00261020 Nervous System 396 NB-1 456 5.873385 0.813 1.039 0.8920.8527 0.9085 0.934 0.9583 0.9709 1.0441 026_370_00252740 Nervous System370 SK-N-SH 456 6.176056 0.926 0.811 0.891 1.0803 2.0427 1.01 0.8920.9269 0.9864 026_364_00252720 Nervous System 364 SK-N-DZ 456 6.2519740.895 1.012 0.958 1.0963 0.9642 0.976 1.0005 0.9787 0.9774026_370_00258530 Nervous System 370 SK-N-SH 456 6.266144 0.36 0.9331.052 1.0961 1.0754 0.948 0.9798 1.0134 0.9668 026_362_00252710 NervousSystem 362 SK-N-AS 456 6.874316 0.911 1.075 0.916 1.0354 0.9179 0.9391.178 0.9451 0.9362 026_368_00252700 Nervous System 368 MC-IXC 4566.982443 0.084 0.616 1.087 1.1559 1.0678 1.11 1.0784 0.9917 0.9902026_8122_00262490 Nervous System 8122 NB12 456 7.149853 0.063 0.9731.019 1.3067 1.0528 0.999 1.4063 0.9955 0.9632 026_934_00287430 Ovary934 A2780 456 −0.227264 0.302 0.3 0.342 0.3503 0.3758 0.467 0.72390.7489 0.9268 026_1126_00293760 Ovary 1126 OV-90 456 0.856076 0.3230.361 0.384 0.4703 0.5785 0.701 0.8275 0.885 0.873 026_1129_00290660Ovary 1129 TOV-112D 456 2.270257 0.47 0.481 0.592 0.7498 0.7692 0.9250.9217 0.9942 1.1258 026_1220_00291150 Ovary 1220 ES-2 456 2.3978680.584 0.504 0.584 0.5655 0.9851 0.957 1.1602 1.1706 1.0966026_949_00290350 Ovary 949 TYK-nu 456 2.489488 0.511 0.577 0.587 0.57680.6153 0.686 0.7718 1.0687 1.0378 026_925_00290850 Ovary 925 IGROV-1 4562.753994 0.494 0.608 0.578 0.7594 0.7462 0.857 0.9169 0.8966 0.8943026_8244_00314220 Ovary 8244 JHOS-4 456 2.779507 0.63 0.601 0.597 0.63810.7711 0.88 0.955 0.9128 0.9028 026_8279_00293400 Ovary 8279 UWB1.289456 2.893774 0.764 0.625 0.723 0.7182 0.783 1.079 0.9597 1.044 1.0467026_940_00290340 Ovary 940 RMG-I 456 3.11591 0.587 0.604 0.66 0.69950.6871 0.846 0.8531 0.9424 1.0257 26_8238_00304370 Ovary 8238 IOSE-364-456 3.149354 0.505 0.763 0.761 0.7451 0.8102 0.848 1.0558 1.0226 1.0295026_8237_00295840 Ovary 8237 Hey 456 3.317969 0.551 0.783 0.688 0.7760.8484 0.917 1.1038 1.0681 1.1892 026_8260_00292720 Ovary 8260 PEO1 4563.508406 0.648 0.61 0.642 0.7269 0.6891 0.74 1.0027 0.9408 0.9148026_8230_00303100 Ovary 8230 DOV13 456 3.68994 0.636 0.73 0.886 0.8510.9406 0.961 0.9388 0.9681 1.0998 026_8230_00292650 Ovary 8230 DOV13 4563.809459 0.641 0.685 0.768 0.9488 0.8764 0.863 0.9076 0.9226 1.0565026_8092_00295850 Ovary 8092 KURAMOCHI 456 3.819916 0.757 0.729 0.6420.6589 0.7541 0.914 0.9211 1.0035 1.0531 026_8240_00291160 Ovary 8240IOSE-523- 456 3.834885 0.676 0.747 0.827 0.9249 0.9868 1 0.9181 0.97421.0197 026_932_00291140 Ovary 932 EFO-27 456 3.943589 0.65 0.668 0.650.7216 0.7753 0.887 0.9104 0.9136 0.9152 026_8084_00292670 Ovary 8084KGN 456 4.00968 0.776 0.686 0.68 0.804 0.816 0.93 0.911 0.9826 0.9092026_1125_00290571 Ovary 1125 Caov-3 456 4.050855 0.676 0.787 0.855 0.9510.9041 1.029 0.9092 0.8992 1.0107 026_8256_00292920 Ovary 8256 OV-7 4564.064163 0.666 0.844 0.821 0.9099 0.9293 0.961 1.0098 0.9638 0.9379026_8241_00292900 Ovary 8241 IOSE-75-16SV40 456 4.094216 0.698 0.8040.834 0.8672 0.894 1.02 1.0443 1.0711 1.107 026_938_00287450 Ovary 938OAW42 456 4.158942 0.689 0.707 0.728 0.8613 0.7453 0.912 0.7815 1.02581.0515 026_1235_00298501 Ovary 1235 Caov-4 456 4.19878 0.673 0.815 0.8510.7794 0.8482 0.895 1.0046 1.0048 0.9449 026_1221_00293780 Ovary 1221 SW626 456 4.261643 0.636 0.704 0.69 0.6894 0.7014 0.755 0.8172 0.88591.0281 026_933_00295510 Ovary 933 FU-OV-1 456 4.35795 0.921 0.786 0.660.785 0.7855 0.932 1.0797 1.0643 1.0563 026_8259_00292710 Ovary 8259OVK-18 456 4.419332 0.714 0.849 0.926 0.7816 0.8328 1.11 1.0158 0.92411.1619 026_8242_00295400 Ovary 8242 JHOS-2 456 4.46038 0.744 0.845 0.8430.8069 0.9687 1.09 0.9491 1.0683 1.0333 026_1128_00292620 Ovary 1128PA-1 456 4.524728 0.741 0.875 0.809 0.8925 0.9409 1.005 1.0033 1.01111.0682 026_938_00290330 Ovary 938 OAW42 456 4.600882 0.772 0.772 0.8430.9103 0.8456 1.001 1.0305 1.0314 1.0721 026_8148_00292700 Ovary 8148OVCAR-4 456 4.649732 0.715 0.824 0.723 0.6704 0.8391 0.855 0.9034 1.09221.0169 026_8243_00295410 Ovary 8243 JHOS-3 456 4.699022 0.801 0.7580.691 0.7427 0.8184 0.86 1.0104 0.9653 1.0478 026_1130_00308501 Ovary1130 TOV-21G 456 4.743414 0.744 0.838 0.882 0.8952 0.9369 0.922 0.92120.9456 0.9683 026_931_00252940 Ovary 931 EFO-21 456 4.786052 0.749 0.9620.957 1.0202 1.0143 0.983 0.9976 0.9688 0.9523 026_931_00290820 Ovary931 EFO-21 456 4.852802 0.791 0.924 0.954 0.9827 1.0247 0.961 0.93530.9274 0.9237 026_932_00288450 Ovary 932 EFO-27 456 4.890908 0.759 0.7290.783 0.7813 0.828 0.851 0.9362 1.0032 1.0515 026_8258_00291180 Ovary8258 OVCA433 456 4.901177 0.703 0.713 0.753 0.7366 0.79 0.864 0.94680.9244 0.9669 026_928_00290870 Ovary 928 OVCAR-5 456 4.903025 0.7910.718 0.699 0.6913 0.7147 0.8 0.831 0.8925 0.9149 026_8257_00293650Ovary 8257 OVCA420 456 4.98883 0.716 0.738 0.79 0.8055 0.8238 0.8190.8677 0.8812 1.0222 026_8257_00292690 Ovary 8257 OVCA420 456 5.0502680.789 0.822 0.798 0.9932 0.8382 0.876 0.928 0.9213 1.1024026_8239_00292890 Ovary 8239 IOSE-397 456 5.092878 0.842 0.8 0.8660.8796 0.8926 0.921 1.2015 0.9976 1.2125 026_941_00288490 Ovary 941 RKN456 5.095259 0.777 0.883 0.798 0.796 0.8682 1.049 1.195 1.0697 1.0119026_929_00290880 Ovary 929 OVCAR-8 456 5.147098 0.763 0.805 0.822 0.84570.8675 0.888 0.9211 0.9687 1.0047 026_8215_00287660 Ovary 8215 OC-314456 5.38803 0.869 0.99 0.986 1.0451 1.0092 1.01 1.0495 0.9963 1.0021026_939_00287460 Ovary 939 SK-OV-3 456 5.430154 0.806 0.848 0.834 0.80940.8719 0.891 0.9424 0.903 1.1026 026_945_00288480 Ovary 945 OVMIU 4565.797932 0.75 0.737 0.748 0.7949 0.7921 0.844 0.8857 0.9368 0.9866026_948_00291230 Ovary 948 OVTOKO 456 5.89738 0.829 0.912 0.942 0.91050.938 0.909 0.9025 0.9329 0.9575 026_8255_00293640 Ovary 8255 OV-56 4565.989644 0.874 0.894 0.904 0.9159 0.9189 0.911 0.9267 0.9453 0.9277026_937_00288460 Ovary 937 OAW28 456 6.027686 0.859 0.878 0.802 0.85120.7724 0.814 1.0447 0.8649 1.0363 026_1127_00296460 Ovary 1127 NIH:OVCAR-3 456 6.109694 0.886 0.95 0.992 0.9785 0.8788 0.986 0.9619 1.07320.8745 026_947_00263500 Ovary 947 OVKATE 456 6.266319 0.819 0.823 0.8750.8874 0.9093 0.906 0.9175 0.9563 0.9507 026_938_00292910 Ovary 938OAW42 456 6.299032 0.918 0.963 0.958 0.9614 0.9778 1.041 1.0213 0.98991.0087 026_945_00256180 Ovary 945 OVMIU 456 6.360298 0.891 0.987 1.0010.9225 0.8452 0.776 0.8273 0.9233 1.0102 026_946_00291190 Ovary 946OVISE 456 6.600815 0.805 0.739 0.737 0.7358 0.7593 0.759 0.8539 0.86640.8624 026_8254_00295890 Ovary 8254 OV-17R 456 6.877329 0.913 1.0410.974 1.0282 1.0077 1.015 1.0376 0.9484 1.0604 026_973_00295540 Pancreas973 HUP-T4 456 0.866739 0.321 0.315 0.362 0.4721 0.5603 0.814 1.09840.8938 1.1407 026_983_00295450 Pancreas 983 SUIT-2 456 1.197889 0.3970.339 0.382 0.581 0.6565 0.942 0.9937 1.019 1.1926 026_982_00292940Pancreas 982 QGP-1 456 1.430625 0.538 0.437 0.492 0.5682 0.6307 0.80.8372 0.9601 1.0476 026_8118_00295430 Pancreas 8118 MZ1-PC 456 1.481130.363 0.489 0.495 0.5335 0.6471 0.671 0.9118 1.008 0.9838026_8149_00293771 Pancreas 8149 PSN1 456 1.829333 0.531 0.553 0.5 0.52310.6104 0.71 0.8375 0.909 0.9509 026_953_00295470 Pancreas 953 AsPC-1 4561.847893 0.476 0.515 0.508 0.4987 0.5741 0.746 0.8281 1.0377 1.0843026_1256_00260300 Pancreas 1256 950-MPS 456 1.902786 0.332 0.473 0.5150.7193 0.8035 0.916 0.9125 1.0441 0.9009 026_976_00298480 Pancreas 976Panc 04.03 456 2.685969 0.592 0.594 0.57 0.562 0.6645 0.706 0.83190.9296 1.0176 026_954_00292870 Pancreas 954 BxPC-3 456 2.90077 0.5740.607 0.558 0.6572 0.7404 0.813 1.0541 1.0916 0.9092 026_967_00292570Pancreas 967 Capan-1 456 3.140577 0.522 0.69 0.758 0.8527 0.6713 0.8740.9506 0.894 0.958 026_975_00295591 Pancreas 975 YAPC 456 3.507979 0.6130.705 0.797 0.9196 0.8833 0.921 1.0616 1.0323 1.0675 026_1135_00292930Pancreas 1135 PL18 456 3.513426 0.636 0.623 0.694 0.6978 0.7917 0.8320.9096 0.9334 0.9721 026_977_00308210 Pancreas 977 KP-1N 456 3.6577780.607 0.764 0.684 0.7525 0.7655 0.908 0.9944 0.9837 0.9553026_969_00295580 Pancreas 969 PA-TU-8988T 456 3.657963 0.664 0.653 0.7250.7145 0.7879 0.992 1.0299 1.0048 1.0863 026_1491_00273490 Pancreas 1491SNU-324 456 3.732679 0.387 0.683 0.791 0.7879 0.8486 0.832 0.9455 0.98130.9695 026_961_00295570 Pancreas 961 Panc 02.03 456 4.088522 0.702 0.6990.633 0.701 0.7003 0.795 0.8748 0.9019 1.1113 026_974_00292601 Pancreas974 HUP-T3 456 4.280085 0.684 0.754 0.793 0.909 0.7596 0.88 0.98950.9696 1.0099 026_963_00293710 Pancreas 963 Hs 766T 456 4.34359 0.7090.746 0.833 0.7623 0.8169 1.052 0.9605 0.9551 1.0544 026_959_00292630Pancreas 959 Panc 03.27 456 4.482408 0.701 0.682 0.805 0.8272 0.75650.838 0.9087 0.9434 1.0258 026_981_00293750 Pancreas 981 KP-4 4564.755774 0.733 0.9 0.784 0.9218 0.8875 1.007 0.9325 1.0003 0.8788026_968_00292580 Pancreas 968 CFPAC-1 456 4.814885 0.772 0.822 0.870.9195 0.9082 0.935 0.8889 1.0106 1.1286 026_1134_00300280 Pancreas 1134PL4 456 4.852316 0.738 0.739 0.727 0.7443 0.7816 0.797 0.9521 0.96631.0376 026_979_00293740 Pancreas 979 KP-3 456 4.900084 0.956 0.777 0.8390.8825 0.8836 0.909 0.9593 0.9092 0.9686 026_956_00292591 Pancreas 956HPAF-II 456 5.005523 0.734 0.786 0.762 0.6523 0.7269 0.755 0.9185 0.9791.0195 026_953_00257150 Pancreas 953 AsPC-1 456 5.141432 0.761 0.7080.731 0.7295 0.7369 0.79 0.8534 0.8911 0.9577 26_968_00304350 Pancreas968 CFPAC-1 456 5.151366 0.817 0.828 0.864 0.8724 0.8715 1.008 1.0070.9779 1.001 026_957_00296350 Pancreas 957 SW 1990 456 5.575766 0.8490.911 0.906 0.8954 0.9494 0.957 0.977 0.9769 1.1173 026_960_00296311Pancreas 960 Panc 08.13 456 5.65459 0.875 0.894 0.924 0.9669 0.9243 0.961.1005 1.065 1.0309 026_951_00256230 Pancreas 951 HPAC 456 5.7363360.862 1.024 0.931 0.8532 0.9386 1.035 1.0328 1.0507 1.0612026_964_00295480 Pancreas 964 Capan-2 456 5.74256 0.877 0.928 0.7590.8346 0.9905 0.925 1.0364 1.027 0.9499 026_1134_00298560 Pancreas 1134PL4 456 5.771259 0.844 0.812 0.91 0.8479 0.7738 0.977 0.9068 1.0590.9774 026_970_00293370 Pancreas 970 PA-TU-8902 456 5.772043 0.841 0.970.919 0.8896 0.9177 0.966 0.9181 1.322 0.9871 026_952_00292610 Pancreas952 MIA PaCa-2 456 5.946631 0.923 0.885 0.718 0.8919 0.8505 1.058 1.05860.8995 1.1403 026_972_00296250 Pancreas 972 DAN-G 456 5.955955 0.8970.924 0.945 0.9646 0.9703 0.978 1.0074 1.032 1.0585 026_951_00295520Pancreas 951 HPAC 456 5.962773 0.923 0.889 0.787 0.9719 0.9562 1.1180.986 0.9708 1.1145 026_975_00252910 Pancreas 975 YAPC 456 5.9898831.015 0.914 0.897 0.8713 0.8903 1.037 0.9777 1.1409 0.981026_963_00252950 Pancreas 963 Hs 766T 456 6.264463 0.496 0.974 0.9531.0445 0.9118 1.028 0.9324 1.0245 1.0186 026_958_00296320 Pancreas 958Panc 10.05 456 6.318194 0.851 0.835 0.851 0.824 0.8929 0.879 0.90031.0065 1.0291 026_955_00292640 Pancreas 955 SU.86.86 456 6.593699 1.0170.82 0.986 0.9378 1.1258 1.072 1.0873 1.0381 0.9789 026_759_00300240Pleura 759 MSTO-211H 456 3.066599 0.666 0.637 0.664 0.6954 0.8026 0.9011.0485 1.0165 1.0436 026_1213_00303050 Pleura 1213 H2818 456 3.1574150.577 0.674 0.728 0.8558 0.9032 0.962 0.9285 1.0744 1.1008026_8116_00303080 Pleura 8116 MPP-89 456 3.723631 0.416 0.808 0.8230.9733 0.9887 0.968 1.0012 0.9912 1.0294 026_1206_00302600 Pleura 1206H2722 456 3.764667 0.604 0.768 0.801 0.8049 0.8692 0.937 0.9073 0.98420.9698 026_1210_00302610 Pleura 1210 H2803 456 4.117283 0.686 0.7230.669 0.7081 0.719 0.892 0.9533 1.0129 0.9547 026_1215_00311260 Pleura1215 H290 456 4.122283 0.629 0.753 0.69 0.7652 0.7781 0.737 0.81990.9853 0.9812 026_1206_00308200 Pleura 1206 H2722 456 4.184817 0.6990.769 0.85 0.8838 0.889 0.906 1.0135 0.9948 1.0129 026_1212_00300580Pleura 1212 H2810 456 4.445211 0.69 0.929 0.942 0.9243 0.9927 0.8840.9289 0.9051 0.9626 026_682_00304600 Pleura 682 NCI-H2452 456 4.4524680.722 0.847 0.891 0.906 0.9407 0.899 0.9567 1.0786 1.1287026_1200_00300690 Pleura 1200 H2373 456 4.612295 0.695 0.911 0.8870.9257 0.9258 0.849 0.9218 0.9772 0.9399 026_1214_00308460 Pleura 1214H2869 456 4.934927 0.775 0.881 0.878 0.926 0.9241 0.914 0.9475 0.95361.0138 026_1214_00308790 Pleura 1214 H2869 456 4.961713 0.802 0.8790.908 0.9132 0.9528 0.991 0.9959 1.0132 1.0202 026_1202_00303040 Pleura1202 H2591 456 5.067997 0.82 0.918 0.945 1.0327 0.9338 0.983 0.95411.0755 1.1707 026_1209_00300210 Pleura 1209 H28 456 5.341766 0.817 0.8210.827 0.8331 0.8999 0.927 0.9491 1.0072 0.9857 026_8078_00303060 Pleura8078 IST-MES1 456 5.358253 0.782 0.897 0.859 0.8822 0.8445 0.907 0.97520.926 1.0513 026_1199_00300560 Pleura 1199 H2369 456 5.439636 0.82 0.910.99 0.8596 0.9181 0.984 0.9172 0.9896 1.1394 026_8078_00304970 Pleura8078 IST-MES1 456 5.505825 0.847 0.88 0.853 0.8587 0.8938 0.89 0.94160.9423 1.0968 026_1207_00298510 Pleura 1207 H2731 456 5.56988 0.8191.018 1.003 0.8986 0.9778 0.921 0.9543 0.9856 0.9695 026_1198_00302580Pleura 1198 H2052 456 5.579483 0.859 0.966 0.995 0.9551 0.9651 1.011.0265 1.0233 1.0095 026_1201_00302590 Pleura 1201 H2461 456 5.6541170.856 0.868 0.914 0.904 0.8852 0.908 0.9343 1.0239 1.1079026_1213_00300720 Pleura 1213 H2818 456 5.828097 0.843 0.946 0.86 0.96820.9236 0.942 0.9493 0.9688 0.9576 026_1211_00300570 Pleura 1211 H2804456 5.890027 0.881 0.886 0.907 0.8753 0.9153 0.955 0.969 0.9857 1.0448026_1218_00300730 Pleura 1218 H513 456 5.953268 0.9 0.905 0.905 1.07221.0284 0.976 0.9507 1.0341 1.2023 026_1208_00300710 Pleura 1208 H2795456 6.063798 0.3 0.943 0.961 0.8752 0.9823 0.905 0.9466 0.9643 0.975026_1203_00300850 Pleura 1203 H2595 456 6.184806 0.92 0.909 0.899 1.03571.0632 0.905 0.974 1.018 0.9037 026_8245_00282710 pleural effusion 8245KMS-11 456 0.702427 0.356 0.381 0.404 0.4804 0.491 0.652 0.7128 1.06451.1048 026_996_00298490 Prostate 996 22RV1 456 2.346887 0.542 0.5210.592 0.6234 0.6635 0.707 0.7764 0.8517 0.9168 026_985_00303070 Prostate985 LNCaP clone FGC 456 4.018762 0.727 0.863 0.923 1.1164 1.0922 0.9841.0179 1.1749 1.0533 026_987_00298550 Prostate 987 PC-3 456 4.0976960.698 0.761 0.871 0.9338 0.9109 0.914 0.9938 1.2109 0.991026_1001_00300200 Prostate 1001 DU 145 456 5.044916 0.768 0.832 0.8670.8699 0.8879 0.906 0.9933 0.9168 1.1136 026_988_00308270 Prostate 988PWR-1E 456 5.228199 0.804 0.854 0.877 0.8566 0.8621 0.923 0.9727 0.95160.9806 026_997_00300660 Prostate 997 BPH-1 456 5.448713 0.479 0.8681.201 1.1209 1.1016 0.674 0.9562 1.1603 0.8983 026_1000_00300740Prostate 1000 VCaP 456 5.968581 0.692 1.071 1.005 1.0988 0.9203 0.910.9225 0.9154 1.1301 026_1009_00264700 Skin 1009 WM35 456 −2.3725120.112 0.201 0.203 0.2168 0.2138 0.242 0.3676 0.463 0.7025026_8212_00264590 Skin 8212 CP50-MEL-B 456 −1.651586 0.151 0.398 0.3920.3817 0.3556 0.35 0.4631 0.5633 0.8209 026_1039_00265170 Skin 1039SK-MEL-30 456 −1.476493 0.263 0.352 0.317 0.3236 0.3515 0.424 0.55340.8565 0.8549 026_1023_00269690 Skin 1023 SK-MEL-2 456 −1.357658 0.1470.207 0.235 0.2621 0.2935 0.343 0.5053 0.6866 0.8939 026_1034_00264680Skin 1034 MEL-HO 456 −0.964561 0.144 0.372 0.375 0.3139 0.3537 0.3270.4189 0.6188 0.8613 026_8073_00263480 Skin 8073 HT-144 456 −0.6412330.154 0.241 0.236 0.3036 0.3695 0.397 0.6432 0.7444 0.9033026_8114_00259970 Skin 8114 MMAC-SF 456 −0.613008 0.211 0.399 0.4920.3813 0.4238 0.662 0.5683 0.6942 1.0563 026_8209_00266540 Skin 8209A4-Fuk 456 −0.557176 0.179 0.476 0.467 0.4234 0.4176 0.498 0.6644 0.73380.7609 026_1046_00263470 Skin 1046 HMVII 456 −0.454742 0.389 0.419 0.4030.3903 0.4097 0.503 0.6117 0.8285 1.0625 026_8120_00262840 Skin 8120MZ7-mel 456 −0.254795 0.073 0.323 0.41 0.4001 0.4106 0.468 0.6687 0.63110.8761 026_8191_00260580 Skin 8191 UACC-257 456 −0.10266 0.456 0.4680.426 0.404 0.4006 0.534 0.6654 0.7954 0.894 026_1176_00266430 Skin 1176451Lu 456 −0.00066 0.26 0.428 0.38 0.386 0.4254 0.499 0.6795 0.84230.9729 026_1149_00263460 Skin 1149 G-MEL 456 0.018696 0.065 0.23 0.1810.1928 0.2465 0.404 0.7707 0.8991 0.9576 026_1147_00263750 Skin 1147SK-MEL-28 456 0.142676 0.187 0.48 0.376 0.4566 0.4611 0.565 0.60830.8116 0.9418 026_1037_00260080 Skin 1037 SK-MEL-1 456 0.168848 0.1760.417 0.368 0.3716 0.3919 0.613 0.6978 0.9091 0.966 026_1190_00266570Skin 1190 Hs 939.T 456 0.185332 0.283 0.631 0.615 0.5802 0.5603 0.5590.5576 0.7187 0.8082 026_1024_00265150 Skin 1024 M-14 456 0.192152 0.4180.433 0.383 0.391 0.4651 0.618 0.742 0.7678 0.9177 026_1025_00269710Skin 1025 COLO-679 456 0.228168 0.267 0.288 0.296 0.3664 0.4611 0.6810.8141 0.9334 1.1196 026_8161_00263740 Skin 8161 SH-4 456 0.433257 0.2030.383 0.33 0.3147 0.3323 0.488 0.7539 0.8959 0.983 026_8097_00262820Skin 8097 LB2518-MEL 456 0.437866 0.076 0.501 0.37 0.3843 0.4237 0.7060.7414 0.8914 0.9396 026_8120_00260550 Skin 8120 MZ7-mel 456 0.4477060.299 0.428 0.435 0.4782 0.4839 0.498 0.6521 0.7326 0.8788026_1033_00262470 Skin 1033 IPC-298 456 0.460127 0.363 0.36 0.404 0.42970.5245 0.562 0.7868 0.9191 0.8529 026_1031_00264030 Skin 1031 IGR-37 4560.57267 0.159 0.498 0.512 0.4794 0.5367 0.602 0.8381 0.9754 1.0032026_1006_00260110 Skin 1006 WM-115 456 0.616077 0.079 0.437 0.583 0.49120.4928 0.627 0.6176 0.6485 0.9586 026_8023_00265291 Skin 8023 COLO-829456 0.740747 0.164 0.659 0.533 0.5534 0.5976 0.709 0.8699 0.9148 0.9877026_1036_00264690 Skin 1036 RVH-421 456 0.784255 0.099 0.511 0.4890.5329 0.5785 0.531 0.5954 0.5867 0.7408 026_1011_00269770 Skin 1011WM278 456 0.944573 0.289 0.463 0.457 0.4545 0.4605 0.568 0.7513 0.88360.9597 026_1003_00269650 Skin 1003 G-361 456 0.948854 0.099 0.326 0.4340.5233 0.6155 0.705 0.83 0.8998 1.0756 026_8002_00263700 Skin 8002 A101D456 0.960267 0.088 0.468 0.524 0.5105 0.5276 0.55 0.5835 0.7643 0.8791026_8104_00260540 Skin 8104 LOXIMVI 456 1.072404 0.278 0.403 0.39 0.47690.6158 0.839 0.877 0.864 0.9209 026_1005_00268780 Skin 1005 A-375 4561.191442 0.45 0.499 0.485 0.4604 0.5149 0.506 0.6624 0.892 0.928026_1004_00260870 Skin 1004 C32 456 1.195832 0.288 0.331 0.256 0.2650.3055 0.504 0.8497 0.9278 0.9753 026_1030_00264020 Skin 1030 IGR-1 4561.307792 0.075 0.388 0.556 0.5173 0.6922 0.688 0.8911 0.7819 0.8658026_1035_00265160 Skin 1035 MEL-JUSO 456 1.414781 0.305 0.481 0.4940.5153 0.7013 0.739 0.9127 0.9597 1.0093 026_8119_00259980 Skin 8119MZ2-MEL. 456 1.446447 0.402 0.467 0.438 0.4246 0.4152 0.679 0.73450.8985 0.9484 026_8097_00260530 Skin 8097 LB2518-MEL 456 1.447504 0.4390.438 0.44 0.3578 0.3688 0.635 0.8111 0.8622 0.8731 026_8225_00264640Skin 8225 SK-MEL-5 456 1.574212 0.07 0.432 0.581 0.5597 0.6534 0.6030.7648 0.9285 1.0194 026_8104_00262830 Skin 8104 LOXIMVI 456 1.6764150.172 0.565 0.594 0.5546 0.7085 0.865 1.2098 1.0454 1.1061026_1042_00274340 Skin 1042 COLO 792 456 1.75025 0.329 0.514 0.5730.6166 0.6518 0.787 0.8993 0.9066 0.9485 026_1026_00269720 Skin 1026COLO-783 456 1.791757 0.479 0.599 0.603 0.6144 0.5728 0.634 0.69790.8575 0.9077 026_1010_00262530 Skin 1010 WM1552C 456 2.109919 0.3640.794 0.685 0.6806 0.6737 0.881 0.8586 0.9992 0.9359 026_1041_00263450Skin 1041 A431 456 2.481335 0.436 0.563 0.633 0.821 0.8517 0.858 1.07920.8962 0.9346 026_8098_00269090 Skin 8098 LB373-MEL-D 456 2.623655 0.4960.615 0.627 0.6225 0.6708 0.815 0.8109 0.9711 1.0201 026_1022_00262940Skin 1022 RPMI-7951 456 2.647594 0.249 0.552 0.678 0.6664 0.7181 0.8240.8966 0.8605 0.908 026_1181_00269080 Skin 1181 Hs 944.T 456 2.8242830.206 0.612 0.779 0.8499 0.8608 0.892 0.9924 1.1546 1.1214026_1008_00262540 Skin 1008 WM793B 456 2.933974 0.371 0.744 0.693 0.69620.7393 0.731 0.8508 0.9586 0.8942 026_1027_00265280 Skin 1027 COLO-800456 3.132083 0.537 0.717 0.804 0.8959 0.9417 0.926 0.9678 0.954 1.0136026_8034_00265300 Skin 8034 DJM-1 456 3.234358 0.554 0.778 0.806 0.94520.9331 1.005 0.9966 1.0061 1.0084 026_1181_00264620 Skin 1181 Hs 944.T456 3.491747 0.094 0.698 0.822 0.9152 0.8448 0.9 0.9921 0.9743 0.9841026_8225_00263510 Skin 8225 SK-MEL-5 456 3.505326 0.234 0.653 0.6940.7331 0.7681 0.79 0.9216 0.9437 0.9528 026_1145_00263720 Skin 1145CHL-1 456 3.811805 0.609 0.895 0.833 0.8778 0.9025 0.855 0.9286 0.93161.0458 026_8060_00265311 Skin 8060 GAK 456 4.2621 0.37 0.758 0.8270.7891 0.8733 0.894 0.9899 0.9692 0.9911 026_1047_00265320 Skin 1047MEWO 456 4.342961 0.779 0.931 0.98 0.9757 0.9449 0.991 1.0008 0.9996 1026_1022_00260910 Skin 1022 RPMI-7951 456 4.58823 0.257 0.748 0.8010.8703 0.8751 0.884 0.9495 0.9173 0.9542 026_1038_00260090 Skin 1038SK-MEL-3 456 4.834702 0.151 0.731 0.777 0.7955 0.7955 0.827 0.8793 0.9130.9361 026_1002_00260861 Skin 1002 A2058 456 5.928358 0.795 0.897 0.850.894 0.8474 0.885 1.0053 0.8902 0.984 026_8025_00306491 Skin 8025CP66-MEL 456 6.278644 0.914 0.888 1.059 0.9053 1.0334 0.961 0.87981.0258 0.9475 026_1120_00264061 Skin 1120 UACC-62 456 6.317961 0.4170.99 0.941 0.9531 0.9635 1.005 1.0535 1.0033 0.9903 026_1049_00269761Skin 1049 VMRC-MELG 456 6.363031 0.904 0.991 0.949 0.974 0.9849 1.0041.032 1.0282 1.0264 026_8077_00311281 Skin 8077 IST-MEL1 456 6.4862150.817 0.849 0.783 0.8212 0.8361 0.894 0.8282 0.9342 0.9134026_1002_00262871 Skin 1002 A2058 456 6.768535 0.494 1.023 0.918 0.99570.9334 0.899 0.9223 0.931 0.9502 026_1191_00266580 Skin 1191 Hs 940.T456 7.017835 0.562 0.98 1.123 1.1931 0.9727 1.159 1.1334 1.0446 1.0643026_8211_00306531 Skin 8211 SK-MEL-24 456 7.240348 0.995 1.062 1.0470.9379 1.0364 1.075 1.0521 1.0504 1.0137 026_1004_00262880 Skin 1004 C32456 7.407136 1.327 0.196 1.169 0.1356 1.1547 0.213 1.1935 0.7281 1.1468026_8077_00263490 Skin 8077 IST-MEL1 456 8.189893 1.25 1.21 1.184 1.15650.9453 0.931 1.1642 1.0861 1.0387 026_1076_00260350 Stomach 1076 OCUM-1456 −1.975727 0.138 0.299 0.203 0.1441 0.1472 0.137 0.1598 0.2644 0.5747026_1050_00262790 Stomach 1050 AGS 456 0.402284 0.198 0.304 0.337 0.44110.6201 0.452 0.6355 0.7729 1.0239 026_1070_00258920 Stomach 1070 HSC-39456 1.218484 0.163 0.38 0.469 0.5806 0.7048 0.892 1.16 0.922 1.1787026_8193_00311240 Stomach 8193 ECC10 456 1.328742 0.306 0.42 0.4450.5445 0.7218 0.758 0.8813 0.9883 0.9993 026_1052_00255810 Stomach 1052SNU-1 456 1.425654 0.369 0.469 0.497 0.5342 0.5814 0.77 0.7984 0.82850.947 026_1056_00316590 Stomach 1056 KATO III 456 1.949149 0.309 0.5310.622 0.6777 0.7504 0.912 0.9052 1.0407 1.0158 026_1060_00256830 Stomach1060 MKN45 456 2.285447 0.504 0.586 0.532 0.5224 0.5043 0.577 0.77570.8156 0.9028 026_1072_00258880 Stomach 1072 23132/87 456 2.907608 0.4730.717 0.911 0.8748 1.0846 1.055 1.07 0.981 1.001 026_1060_00262910Stomach 1060 MKN45 456 2.913103 0.498 0.498 0.474 0.9422 0.5296 0.6231.0575 0.9958 0.8867 026_1078_00263960 Stomach 1078 IM-95 456 3.2096610.348 0.625 0.723 1.042 0.7453 0.866 0.8746 0.9835 0.9734026_1054_00308870 Stomach 1054 SNU-16 456 3.34225 0.574 0.78 0.8830.9578 0.9599 0.973 0.9861 1.0293 1.027 026_1064_00258960 Stomach 1064NUGC-3 456 3.516892 0.253 0.754 0.947 0.9636 0.9596 1.019 1.0426 1.02781.0027 026_1075_00308260 Stomach 1075 NUGC-4 456 3.652276 0.617 0.8270.703 0.9984 1.0102 1.068 1.1379 0.9929 1.1512 026_1065_00273570 Stomach1065 MKN7 456 3.792655 0.621 0.777 0.872 0.891 0.9122 0.904 0.91970.9359 0.9547 026_1067_00311560 Stomach 1067 RERF-GC-1B 456 4.1661680.732 0.677 0.761 0.8438 0.781 0.877 0.8716 0.958 1.077226_1067_00314080 Stomach 1067 RERF-GC-1B 456 4.402339 0.708 0.833 0.8310.8383 0.8843 0.854 0.9212 0.9276 1.1227 026_1068_0028930 Stomach 1068MKN28 456 4.480011 0.554 0.885 0.897 0.9579 0.9774 1.009 1.0156 1.00421.0326 026_1057_00271310 Stomach 1057 Hs 746T 456 4.481195 0.745 0.7430.677 0.6977 0.6882 0.86 0.875 0.9503 1.0984 026_1060_00260890 Stomach1060 MKN45 456 4.604315 0.753 0.738 0.731 0.6895 0.6891 0.836 1.07271.0008 0.9928 026_1051_00269200 Stomach 1051 FU97 456 4.755599 0.7380.851 0.914 0.8985 0.8947 0.955 0.9241 0.9866 1.0457 026_8187_00316461Stomach 8187 TGBC11TKB 456 4.931222 0.775 0.953 0.925 0.9227 0.94880.988 0.9649 0.9982 0.9763 026_1058_00263730 Stomach 1058 NCI-N87 4565.400356 0.754 0.824 0.808 0.8382 0.8486 0.868 0.9105 0.9132 0.9828026_1064_00264040 Stomach 1064 NUGC-3 456 6.53754 0.915 1.021 1.0071.0316 0.9966 1.014 1.0431 0.9819 1.0068 026_1077_00264820 Stomach 1077SCH 456 6.77844 0.466 1.271 1.195 1.2103 1.1255 0.977 0.9179 1.13151.1153 026_1051_00265140 Stomach 1051 FU97 456 6.812551 0.149 0.7520.783 0.8562 0.833 0.758 0.8748 0.8287 0.8438 026_8067_00258320 Stomach8067 GT3TKB 456 7.007727 0.623 0.977 1.004 1.0575 1.0443 1.026 1.05981.0143 1.0267 026_1053_00258360 Stomach 1053 SNU-5 456 7.265878 1 0.9670.868 0.95 0.958 1.018 1.0763 1.0284 1.0105 026_1073_00258910 Stomach1073 HGC-27 456 7.474785 0.055 1.003 1.012 1.0288 1.0225 1.013 1.03310.9845 0.9972 026_8062_00266170 Stomach 8062 GCIY 456 7.70033 1.0761.218 0.962 1.0422 0.9155 1.202 0.9466 0.9302 1.1001 026_1059_00256820Stomach 1059 MKN1 456 7.84127 1.023 1.223 1.097 1.1806 1.0334 0.9241.0209 0.9691 0.9885 026_8216_00258330 Stomach 8216 RF-48 456 8.0411010.627 1.056 1.075 1.0873 1.0084 1.031 1.0144 1.008 0.9914026_8143_00302630 Testes 8143 NTERA-S-c1-D1 456 4.238289 0.709 0.740.781 0.8439 0.8942 0.971 0.9553 0.9264 1.0878 026_1081_00299690 Testes1081 NCC-IT-A3 456 4.69755 0.834 0.925 0.99 1.0022 1.0753 0.959 1.06640.9907 0.9674 026_1082_00300430 Testes 1082 NEC8 456 5.794775 0.5180.919 1.026 1.1263 1.0267 0.882 1.2293 0.8206 0.9481 026_1087_00311210Thyroid 1087 BHT-101 456 0.094631 0.409 0.442 0.405 0.4025 0.4677 0.6060.749 0.9156 0.9734 026_1098_00252970 Thyroid 1098 IHH-4 456 0.7342990.193 0.418 0.365 0.5027 0.4309 0.608 0.6796 0.9903 0.9333026_1093_00253040 Thyroid 1093 TT2609-C02 456 1.438563 0.33 0.462 0.4890.4674 0.6027 0.719 0.8623 0.9552 1.0031 026_1100_00252990 Thyroid 1100KMH-2 456 1.898194 0.142 0.5 0.577 0.6566 0.6074 0.682 0.7694 0.88880.9822 026_1085_00252920 Thyroid 1085 8505C 456 2.3348 0.536 0.498 0.6050.5369 0.584 0.788 0.7985 1.0523 0.937 026_1088_00252930 Thyroid 1088CAL-62 456 2.495611 0.451 0.785 0.606 0.8114 0.6653 0.836 1.0285 1.00530.944 026_1089_00261010 Thyroid 1089 HTC-C3 456 2.538171 0.577 0.550.623 0.7757 0.5886 0.616 0.7161 0.7476 0.9625 026_1090_00311330 Thyroid1090 ML-1 456 2.64388 0.368 0.64 0.793 0.8005 0.8127 0.931 0.9783 0.93891.0355 026_1090_00253010 Thyroid 1090 ML-1 456 3.121258 0.165 0.7780.787 0.6376 0.6432 0.975 1.0524 0.7737 1.0765 026_1090_00308850 Thyroid1090 ML-1 456 3.18393 0.308 0.673 0.724 0.8112 0.8338 0.925 0.90160.8988 1.0407 026_1086_00255730 Thyroid 1086 B-CPAP 456 3.682081 0.1710.695 0.763 0.7585 0.7239 0.856 0.9452 0.9473 1.0079 026_8020_00314210Thyroid 8020 CGTH-W-1 456 4.407018 0.694 0.874 0.871 0.8186 0.9478 0.9650.9497 1.0206 1.0176 026_1084_00266520 Thyroid 1084 8305C 456 4.4421790.592 0.739 0.746 0.7517 0.73 0.793 0.8273 0.8894 0.8901026_8213_00259200 Thyroid 8213 TT 456 4.648028 0.769 0.966 0.965 0.98310.9968 0.984 0.9765 0.9632 0.942 026_8082_00252980 Thyroid 8082 K5 4566.244906 0.27 0.935 0.007 0.9319 0.951 0.93 0.997 0.9506 0.9691026_1099_00306470 Thyroid 1099 ASH-3 456 6.262224 0.912 0.905 0.9640.9293 0.932 0.965 0.9777 0.9947 1.0629 026_1094_00259110 Thyroid 1094FTC-133 456 6.573461 0.084 0.958 0.978 1.0951 1.0879 1.057 1.063 1.02540.8654 026_1092_00259170 Thyroid 1092 S-117 456 6.767861 0.271 0.9730.988 1.0669 1.039 1.047 1.018 1.0563 1.0542 026_1097_00259160 Thyroid1097 RO82-W-1 456 7.698514 0.685 1.079 1.053 1.0554 1.0411 1.049 1.06821.0349 1.0563 026_8036_00304360 Thyroid 8036 DSH1 456 1.200178 0.3770.348 0.397 0.5545 0.7402 0.871 0.9778 0.9884 1.0288 26_24_00298840UrinaryTrack 24 RT4 456 2.281802 0.504 0.543 0.577 0.6272 0.7473 0.8891.0564 1.0628 1.0768 026_18_00299720 UrinaryTrack 18 RT-112 456 2.4483550.479 0.581 0.587 0.6728 0.7768 0.797 0.8705 1.106 1.03 026_24_00252540UrinaryTrack 24 RT4 456 2.668527 0.216 0.59 0.634 0.6994 0.7144 0.8550.9368 0.9696 1.0194 026_8036_00257170 UrinaryTrack 8036 DSH1 4562.728478 0.679 0.605 0.639 0.6941 0.7355 0.829 0.9411 1.0033 1.0113026_9_00298850 UrinaryTrack 9 SW 780 456 2.896276 0.523 0.587 0.6760.7538 0.7859 0.866 0.9594 0.9717 1.0348 026_15_00316530 UrinaryTrack 15BFTC-905 456 3.218144 0.607 0.59 0.688 0.6971 0.8338 0.848 0.8978 1.04481.0799 026_8_00298880 UrinaryTrack 8 UM-UC-3 456 3.221517 0.557 0.720.789 0.8471 0.8599 0.995 1.0484 1.045 1.0264 026_6_00303090UrinaryTrack 6 5637 456 3.348554 0.553 0.725 0.744 0.7649 0.9313 0.9180.9221 0.9563 1.0354 026_6_00256650 UrinaryTrack 6 5637 456 3.8372330.726 0.703 0.715 0.7187 0.7189 0.755 0.9193 0.965 1.0004026_8101_00256280 UrinaryTrack 8101 LB831-BLC 456 3.939929 0.504 0.8350.952 1.0143 1.0014 1.01 0.9848 0.9739 0.9713 026_11_00298870UrinaryTrack 11 T24 456 4.120815 0.697 0.744 0.763 0.8533 0.8788 0.9410.9969 1.0222 1.1105 026_19_00298860 UrinaryTrack 19 SW-1710 4564.713264 0.758 0.847 0.828 0.8254 0.8943 0.979 0.9968 1.0264 1.0955026_7_00302400 UrinaryTrack 7 SCaBER 456 4.780909 0.779 0.775 0.7080.7824 0.8163 0.882 1.0463 0.9494 1.0621 026_8101_00302620 UrinaryTrack8101 LB831-BLC 456 4.856105 0.76 0.882 0.929 0.9235 0.9393 0.926 0.93860.9281 1.0507 026_22_00298810 UrinaryTrack 22 HT 1376 456 4.888331 0.8230.879 1.047 0.8692 1.0283 0.988 1.0731 1.1432 1.097 026_20_00299740UrinaryTrack 20 VM-CUB1 456 4.987745 0.751 0.753 0.758 0.8989 0.87260.877 0.9581 0.9471 0.9762 026_14_00298780 UrinaryTrack 14 647-V 4565.049696 0.854 0.851 0.873 0.8859 0.9584 1.097 1.1389 1.2074 1.0083026_12_00299730 UrinaryTrack 12 TCCSUP 456 5.306108 0.821 0.876 0.8970.9178 0.9315 0.962 0.9176 1.0469 1.0217 026_13_00298770 UrinaryTrack 13639-V 456 5.658711 0.867 0.933 0.849 0.9317 0.8466 1.046 1.0223 1.12661.2765 026_16_00298800 UrinaryTrack 16 CAL-29 456 5.956916 0.811 0.8340.909 0.8884 0.9073 0.924 0.956 0.9709 0.9841 026_3_00308800UrinaryTrack 3 HT-1197 456 6.060787 0.877 0.935 0.984 0.8762 0.9541 0.871.0393 1.0266 1.0429 026_17_00300420 UrinaryTrack 17 KU-19-19 4566.388593 0.216 0.953 0.975 0.9996 0.9834 0.744 0.9959 0.9777 1.0473026_10_00298820 UrinaryTrack 10 J82 456 6.755266 0.951 0.965 0.9250.9536 1.0393 1.06 1.0882 1.0657 1.037 026_8154_00262510 Uterus 8154RL95-2 456 0.033885 0.116 0.241 0.307 0.315 0.5516 0.413 0.6863 0.8140.8778 026_1116_00268860 Uterus 1116 SNG-M 456 2.236003 0.377 0.6050.635 0.4653 0.7155 0.805 0.9708 1.0702 1.1724 026_8154_00280250 Uterus8154 RL95-2 456 2.655708 0.475 0.611 0.69 0.5902 0.6865 0.677 0.8260.8426 0.9723 026_8166_00269750 Uterus 8166 SK-UT-1 456 2.769805 0.3990.642 0.861 0.9305 0.8691 0.946 0.8217 0.867 0.9308 026_1115_00268850Uterus 1115 SKN 456 3.232484 0.402 0.724 0.819 0.8345 0.9291 0.9411.0001 0.9878 0.9814 026_1112_00268820 Uterus 1112 Ishikawa 456 3.5728440.62 0.659 0.62 0.6814 0.702 0.72 0.9036 0.8807 1.0744 (Heraldio) 02 ER-026_1107_00311310 Uterus 1107 MFE-296 456 3.590101 0.601 0.697 0.6470.7074 0.7773 0.802 0.8186 0.9166 1.0105 026_1108_00308840 Uterus 1108MFE-319 456 3.953038 0.7 0.723 0.698 0.8033 0.8993 0.819 0.9773 1.11891.0369 026_1109_00269701 Uterus 1109 COLO 684 456 4.059524 0.645 0.8180.925 0.873 0.8541 0.934 0.9505 0.9782 1.0023 026_1102_00268790 Uterus1102 AN3CA 456 4.238238 0.395 0.808 0.815 0.8203 0.9291 0.817 0.97860.9815 0.9877 026_1108_00314330 Uterus 1108 MFE-319 456 4.375739 0.7450.784 0.794 0.8526 0.9259 0.907 1.0498 1.0643 1.1339 026_1113_00268840Uterus 1113 MES-SA 456 4.83984 0.832 0.962 1.003 0.9833 0.9906 0.9840.9981 1.0121 0.9684 026_1117_00268810 Uterus 1117 HEC-1 456 5.2150290.824 0.904 0.885 0.8856 0.9834 0.972 0.9217 1.0697 1.0963026_1113_00318661 Uterus 1113 MES-SA 456 5.307659 0.807 0.995 0.9470.9632 1.0122 1.033 1.0384 1.0103 0.9636 026_8206_00264270 Uterus 8206KLE 456 5.888467 0.885 1 1.029 1.0338 1.0454 1.029 0.9964 1.0291 0.9684026_1105_00308190 Uterus 1105 ESS-1 456 6.082924 0.887 0.915 0.9110.9349 0.8927 0.94 0.9007 0.9407 0.9198 026_1104_00306500 Uterus 1104 EN456 7.302741 1.047 0.983 0.999 0.9204 1.0053 1.011 1.0162 1.0749 1.0308026_1106_00306510 Uterus 1106 MFE-280 456 7.686964 1.124 0.983 1 0.96831.0601 1.156 1.0799 1.0206 0.9018 026_8163_00302640 Vulva 8163 SK-LMS-1456 2.4327011 0.479 0.526 0.621 0.7198 0.8786 0.991 1.0278 1.0369 1.1613026_8173_00302660 Vulva 8173 SW954 456 2.875791 0.539 0.622 0.644 0.69560.8294 0.866 1.072 1.0089 1.087 26_8174_00304380 Vulva 8174 SW962 4564.641057 0.756 0.704 0.824 0.7868 0.8056 0.875 0.851 0.96 1.0607026_481_00302570 Vulva 481 CAL-39 456 5.164144 0.803 0.856 0.841 0.88640.9389 0.924 0.983 0.9909 1.1127 026_8174_00306540 Vulva 8174 SW962 4566.896884 0.97 0.963 1.002 0.9267 1.0771 0.973 1.0027 1.0435 1.0588

Example 12 BVD-523 Demonstrates In Vivo Antitumor Activity inBRAF^(V600E)-Mutant Cancer Cell Line Xenograft Models

Based on our in vitro findings that BVD-523 reduced proliferation andinduced apoptosis in a concentration-dependent manner, BVD-523 wasadministered by oral gavage to demonstrate its in vivo anti-tumoractivity in models with MAPK/ERK-pathway dependency. Xenograft models ofmelanoma (cell line A375), and colorectal cancer (cell line Colo205),were utilized, both of which harbor a BRAF^(V600E) mutation.

In A375 cell line xenografts, BVD-523 efficacy was compared with thecontrol cytotoxic alkylating agent temozolomide following 14 days oftreatment. BVD-523 demonstrated significant dose-dependent antitumoractivity starting at 50 mg/kg twice daily (BID) (FIG. 31A). Doses of 50and 100 mg/kg BID significantly attenuated tumor growth, with tumorgrowth inhibition (TGI) of 71% (P=0.004) and 99% (P<0.001),respectively. Seven partial regressions (PRs) were noted in the 100mg/kg BID group; no regression responses were noted in any other group.The efficacy observed compared favorably with that of temozolomide,which when administered at 75 and 175 mg/kg resulted in modestdose-dependent TGI of 34% (P>0.05) and 78% (P=0.005), respectively.

Additionally, BVD-523 demonstrated antitumor efficacy in a Colo205 humancolorectal cancer cell line xenograft model (FIG. 31B). BVD-523 againshowed significant dose-dependent tumor regressions at doses of 50, 75,and 100 mg/kg BID, yielding mean tumor regressions T/T_(i) (T=End oftreatment, T_(i)=Treatment initiation) of −48.2%, −77.2%, and −92.3%,respectively (all P<0.0001). Regression was not observed at the lowestdose of BVD-523 (25 mg/kg BID); however, significant tumor growthinhibition, with a T/C (T=Treatment, C=Control) of 25.2% (P<0.0001), wasobserved. Although not well tolerated, the positive controlchemotherapeutic agent irinotecan (CPT-11) showed significant antitumoractivity, inhibiting Colo205 tumor growth with a T/C of 6.4% (P<0.0001).However, even at its maximum tolerated dose in mice, CPT-11 was not aseffective as BVD-523 at doses of 50, 75, or 100 mg/kg BID.

To establish the relationship between pharmacokinetics andpharmacodynamics, BVD-523 plasma concentrations were compared withpERK1/2 levels measured in the tumor by immunohistochemistry andisotope-tagged internal standard mass spectrometry over a 24-hour periodfollowing a single 100 mg/kg oral dose of BVD-523 (FIG. 31C).Phosphorylation of ERK1/2 was low in untreated tumors (0 hours).Following treatment with BVD-523, ERK1/2 phosphorylation steadilyincreased from 1 hour post-dose to maximal levels at 8 hours post-dose,then returned to pre-dose levels by 24 hours. This increase in pERK1/2correlated with BVD-523 drug plasma concentrations. The in vivoobservation of increased pERK1/2 with BVD-523 treatment is consistentwith earlier in vitro findings (FIG. 30D).

Example 13 BVD-523 Results in ERK1/2 Substrate Inhibition DespiteIncreased ERK1/2 Phosphorylation

To examine the effects of BVD-523 on signaling relative to other knownERK1/2 inhibitors (SCH772984, GDC-0994, and Vx-11e) (Morris et al. 2013and Liu et al. 2015), a large-scale reverse phase protein array (RPPA)of approximately 40 proteins was employed in a variety of cell lineswith sensitivity to ERK inhibition. Cell lines with common alterationsin BRAF and RAS were assayed: BRAF^(V600E) mutant lines A375, Colo205,and HT29; KRAS^(G12C)-mutant cell line MIAPACa-2; KRAS^(G13D)-mutantcell line HCT116; and AN3Ca with atypical HRAS^(F82L) mutation. Changesin protein levels are shown as a percentage change from dimethylsulfoxide (DMSO)-treated parental control (FIG. 32A and Table 23). AllERK inhibitors elicited qualitatively similar protein effects, with theexception of phosphorylation of ERK1/2 (pERK1/2 [ERK1/2-T202, -Y204]);SCH7722984 inhibited pERK1/2 in all cell lines, while BVD-523, GDC-0994,and Vx-11e markedly increased pERK1/2. Phospho-p90 RSK (pRSK1) andcyclin D1, which are proximal and distal targets of pERK1/2,respectively, were similarly inhibited by all inhibitors testedregardless of the degree of ERK1/2 phosphorylation (FIG. 32B). Theseindependent findings for BVD-523 are consistent with studies showingthat phosphorylation of ERK1/2 substrates RSK1/2 remained inhibiteddespite dramatically elevated pERK1/2 by Western blots in A375 cells(FIG. 32D), in addition to protein-binding studies demonstrating BVD-523binding and stabilization of pERK1/2 and inactive ERK1/2 (FIG. 29E andFIG. 29F). Therefore, measuring increased pERK1/2 levels could beconsidered as a clinical pharmacodynamic biomarker for BVD-523, whilequantifying inhibition of ERK1/2 targets such as pRSK1 and DUSP6 as wellcould serve a similar purpose.

Additional protein changes are of note in this RPPA dataset (FIG. 32A).Decreased pS6-ribosomal protein appears to be another pharmacodynamicmarker of ERK1/2 inhibition, as evidenced in all cell lines with allcompounds (FIG. 32B). Furthermore, prominent induction of pAKT appearsto be a cell line-dependent observation, where each ERK1/2 inhibitorinduced pAKT in cell lines A375 and AN3CA cells (FIG. 33).Interestingly, the degree of inhibition of survival marker pBAD appearsto differ between compounds, with only modest inhibition of pBAD byGDC-0994 compared with the other ERK1/2 inhibitors tested (FIG. 32A).

Next, how BVD-523 affects cellular localization of ERK1/2 and downstreamtarget pRSK in a BRAF^(V600E)-mutant RKO colorectal cell line (FIG. 32C)was investigated. In resting cells, ERK1/2 localizes to the cytoplasm,and once stimulated pERK1/2 migrates to target organelles, particularlythe nucleus where transcriptional targets are activated (Weinstein etal. 2016). In DMSO-treated control cells, pERK1/2 is evident in bothnuclear and cytoplasmic fractions, which is likely reflective of MAPKpathway activity due to the presence of BRAF^(V600E) in this cell line.Treatment with BVD-523 resulted in elevated pERK1/2 in the nucleus andcytoplasm as well as a modest increase in nuclear total ERK1/2 comparedwith DMSO-treated cells, suggesting that compound-induced stabilizationof pERK1/2 stimulates some nuclear translocation. Despite increasedpERK1/2 in both compartments, pRSK levels are lower in the cytoplasmicand nuclear compartments compared with DMSO control. Comparator MAPKsignaling inhibitors (i.e., trametinib, SCH7722984, dabrafenib)inhibited phosphorylation of ERK1/2 and RSK, as reflected by lowerlevels in the nuclear and cytoplasmic compartments. These data againsuggest that BVD-523-associated increases in pERK1/2 are evident in boththe cytoplasm and nucleus; however, this does not translate toactivation of target substrates. This is consistent with data presentedin FIG. 30D and FIG. 32A.

TABLE 23 % change from DMSO (matched cell line) Avg(S6 Ribo Avg(S6 RiboProt S235 Prot S240 Avg(p90 Avg(bad Avg(4ebp1 Cell Line Treatment 236)244) Avg(Cyclin D1) RSK S380) S112) T70) A375 BVD −95.3 −91.98 −81.45−71 −72.37 −31.82 A375 Vx (Empty) (Empty) −85.46 −65.25 −69.29 23.33A375 GDC −87.61 −80.3 −81.65 −60.74 −55.32 −29.47 A375 SCH −94.71 −91.78−84.05 −72.44 −71.44 −31.75 AN3Ca BVD −43.41 −22.2 −0.69 −28.11 −54.132.17 AN3Ca Vx −18.54 −12.55 −9.59 −28.41 −45.63 16.67 AN3Ca GDC −30.74−23.47 0.34 −29.44 2.53 11.28 AN3Ca SCH −61.99 −35.88 −11.33 −40.26−39.14 61.57 COLO205 BVD −96.15 −97.33 −23.65 −50.84 −31.51 −36.35COLO205 Vx −93.04 −94.89 −39.79 −58.6 −30.28 −43.71 COLO205 GDC −91.19−91.59 −28.02 −57.5 −6.12 −36.69 COLO205 SCH −94.67 −95.09 −36.7 −62.31−29.4 −27.51 HCT116 BVD −96.31 −96.26 −69.62 −31.81 −34.27 4.03 HCT116Vx −90.03 −86.06 −72.72 −33.05 −23.88 10.35 HCT116 GDC −94.82 −95.1−63.59 −22.25 −12.36 20.5 HCT116 SCH −93.86 −91.07 −73.21 −33.7 −31.295.6 HT29 BVD −44.68 −25.67 −37.21 −60.5 −20.66 −41.47 HT29 Vx −32.8−24.35 −35.2 −43.41 −35.62 −2.89 HT29 GDC −41.45 −21.74 −35.69 −30.59−12.98 1.95 HT29 SCH −44.9 −25.73 −36.66 −53.88 −33.9 −40.58 MIAPaca2BVD −79.46 −88.03 −37.9 −35 −30.29 −9.42 MIAPaca2 Vx −63.36 −74.82−33.96 −39.91 −20.85 −15.72 MIAPaca2 GDC −67.9 −75.59 −31.92 −39.09−10.08 −34.01 MIAPaca2 SCH −77.57 −86.61 −39.88 −38.58 −33.07 19.27 %change from DMSO (matched cell line) Avg(p70 S6 Avg(p70 S6 Kinase T389Kinase S371 Avg(Caspase Cell Line Treatment T412) S394) Avg(ERK 1 2)Avg(Akt) Avg(Raptor) 3 CL D175) A375 BVD −23.79 −8.58 −22.91 −14.03−7.77 −8.06 A375 Vx −25.54 −17.32 −5.39 −30.34 −12.7 −9.27 A375 GDC−31.9 −17.34 31.55 −20.7 −14.32 −16.74 A375 SCH −42.73 −28.72 −21.65−23.26 −11.66 −9.87 AN3Ca BVD −14.78 32.26 −9.05 −22.43 −13.82 −10.8AN3Ca Vx 0.56 44.04 −11.27 −24.62 −2.47 −12.7 AN3Ca GDC 26.01 29.09−2.87 −26.04 −8.05 1.55 AN3Ca SCH −16.63 24.56 −9.27 −16.35 −11.09 1.25COLO205 BVD −36.4 −18.11 −18.83 −3.85 −7.14 −3.18 COLO205 Vx −28 −13.64−12.32 −12.51 −0.05 −2.67 COLO205 GDC −32.2 −13.02 −3.33 −11.83 −5.4823.06 COLO205 SCH −30.4 −14.59 −31.87 −10.31 −2.2 14.08 HCT116 BVD−28.11 −16.9 −29.42 4.41 −7.06 −10.11 HCT116 Vx −20.99 −9.89 −24.01−18.15 −4.32 −5.19 HCT116 GDC −24.73 −11.47 −1.9 −6.13 −6.2 −8.36 HCT116SCH −24.63 −12.3 −10.22 −9.86 −9.66 −4.63 HT29 BVD −24.58 −35.94 −44.3−13.41 −8.53 −7.03 HT29 Vx −12.31 −22.86 0.24 −17.84 −6.53 −2.86 HT29GDC −20.86 −25.73 4.66 −10.01 −6.85 −3.44 HT29 SCH −9.55 −20.52 −37−16.93 −12.18 −7.9 MIAPaca2 BVD −39.23 −28.27 −40.33 23.63 21.15 22.35MIAPaca2 Vx −30.66 −30.35 −14.85 −0.15 5.4 6.17 MIAPaca2 GDC −40.99−14.4 −6.88 4.33 22.43 10.47 MIAPaca2 SCH −50.97 −40.47 −23.09 13.4717.66 21.05 % change from DMSO (matched cell line) Avg(mTOR Avg(p70Avg(Raptor Avg(Bcl Cell Line Treatment S2448) Avg(Bad S155) Avg(c Fos)Avg(Rictor) S6 Kinase) S792) 2 T56) A375 BVD −27.87 −21.9 4.39 −20.11−7.6 −10.33 −8.54 A375 Vx −21.66 −6.3 −13.65 −8.16 −6.42 −0.86 −7.53A375 GDC −23.61 −13.31 −12.46 −23.29 −18.11 −15.93 −4.1 A375 SCH −26.17−13.86 −12.51 −22.13 −17.66 −6.89 −19.55 AN3Ca BVD −10.79 0.66 −5.15−4.52 −10.27 −8.47 −12.85 AN3Ca Vx −2.37 4.59 −5.52 0.02 −2.33 0.37−11.73 AN3Ca GDC −2.96 17.31 −0.63 −9.21 −3.67 4.85 1.23 AN3Ca SCH −4.8412.92 −9.18 −10.89 −7.71 −4.03 −10.73 COLO205 BVD −23.51 −18.18 −12.25−5.21 0.14 0.41 −11.84 COLO205 Vx −8.52 −9.72 −19.34 1.65 −3.42 0.2−12.02 COLO205 GDC −7.36 −9.11 −21.33 −5.04 5.83 −9.04 −4.6 COLO205 SCH−9.44 −10.96 −15.17 −19.07 −2.85 −4.17 −6.73 HCT116 BVD −12.78 −30.72−14.08 −13.05 −12.86 −22.04 −8.36 HCT116 Vx −10.12 −15.59 −13.89 1.78−4.45 −11.21 −25 HCT116 GDC −19.33 −19.71 −10.36 −10.98 −9.9 −15.77−11.96 HCT116 SCH −16.05 −22.96 −15.27 −18.5 −14.12 −18.18 −15.89 HT29BVD −20.68 −25.9 −13.48 −18.76 −10.64 −10.48 −8.18 HT29 Vx −13.94 −11.26−8.23 −2.6 −1.72 −2.13 −25.01 HT29 GDC −11.44 −6.7 −12.92 −10.62 −2.83−1.75 1.61 HT29 SCH −22.65 −7.98 −9.26 −13.03 −2.36 −6.38 −17.9 MIAPaca2BVD −11.73 −5.65 −18.44 −11.11 −3.59 5.95 −1.78 MIAPaca2 Vx −5.38 7.38−4.43 −13.24 −9.27 −3.74 −9.86 MIAPaca2 GDC 11.84 8.18 −16.12 −11.25−3.25 2.18 −2.12 MIAPaca2 SCH −11.48 −1.71 −8.99 −15.15 −7.51 4.43 −1.98% change from DMSO (matched cell line) Avg(Bcl2 Avg(mTOR Avg(TuberinAvg(Chk1 Avg(CREB Cell Line Treatment S70) Avg(Bcl 2) Avg(Bax) S2481)TSC2 Y1571) S345) S133) A375 BVD −7.91 −12.55 −6.98 −3.36 −6.32 −7.43−12.65 A375 Vx −4.02 −9.93 −5.05 −2.9 −3.55 0.56 −15.8 A375 GDC 6.3−3.13 3.97 −19.37 −16.74 −18.35 −1.18 A375 SCH −12.91 −18.53 −17.32−15.82 −13.7 −10.59 −1.55 AN3Ca BVD −14.81 −16.83 −10.47 2.75 2.86−13.34 1.03 AN3Ca Vx −14.39 −18.77 −7.89 7.22 3.8 −9.54 0.16 AN3Ca GDC−0.65 7.8 23.83 25.42 25.18 15.46 5.03 AN3Ca SCH −12.42 −5.14 0.15 11.2413.44 14.56 3.64 COLO205 BVD −10.2 −0.13 0.28 −1.33 −3.48 −4.63 −2.67COLO205 Vx −20.82 −14.09 −9.16 −9.43 −9.45 13.12 −10.64 COLO205 GDC −917.95 8.2 0.96 0.66 6.08 −11.85 COLO205 SCH −18.52 −14.56 −6.44 −6.68−1.38 1 −14.56 HCT116 BVD −8.23 −3.59 −0.8 −34.09 −29.89 −39.65 −30.64HCT116 Vx −17.79 −17.3 −20.46 −17.82 −16.51 −16.49 −9.17 HCT116 GDC−12.53 −4.56 −20.11 −18.34 −9.4 −18.77 −8.56 HCT116 SCH −18.34 −10.58−13.86 −22.53 −16.17 −18.51 −24.18 HT29 BVD 2.83 10.68 8.39 −8.64 0.66−13.48 −3.46 HT29 Vx −12.55 −16.73 −18.47 0.52 2.85 6.44 14.16 HT29 GDC10.59 18.16 1.1 3.23 11.86 6.88 9.54 HT29 SCH −11.45 −10.01 −20.59 13.9313.25 19.05 9.87 MIAPaca2 BVD 0.89 5.86 23.44 −0.24 1.7 1.2 −19.26MIAPaca2 Vx −7.83 −2.3 4.33 −3.12 −0.01 2.09 −13.28 MIAPaca2 GDC −6.78−7.8 3.48 3.65 5.71 32.96 −21.37 MIAPaca2 SCH −4.23 4.96 15.67 2.43 8.8613.39 −6.63 % change from DMSO (matched cell line) Avg(Caspase Cell LineTreatment 7 Cl D198) Avg(Stat3) Avg(Bak) Avg(MAK1 S360) Avg(mTOR) Avg(cMyc) Avg(Stat1) Avg(Mcl1) A375 BVD 3.18 6.02 −3.26 3.35 0.2 2.56 1.3210.67 A375 Vx −7.62 1.71 0.65 −7.63 −3.31 −0.97 0.23 1.6 A375 GDC −5.5314.24 2.76 −5.21 −6.2 −9.11 −5.67 −9.09 A375 SCH −6 −1.42 −2.37 0.97−5.18 1.86 −6.01 2.11 AN3Ca BVD −12.35 −4.97 3.38 0.49 5.58 −0.58 −5.65−4.9 AN3Ca Vx −8.09 −7.67 −1.6 6.83 −6.27 −5.47 −0.83 −4.24 AN3Ca GDC−15.56 −13.05 7.33 6.84 −2.85 3.99 9.15 −4.16 AN3Ca SCH −24.17 −6.019.73 −5.02 −5.33 0.88 5.52 −7.15 COLO205 BVD 6.76 −2.58 3.79 14.69 12.77−1.11 0.53 −2.7 COLO205 Vx 0.03 −7.96 −2.79 6.22 3.62 −7.42 −6.1 −10.64COLO205 GDC 1.37 −0.86 6.58 0.82 3.39 −6.22 7.26 2.5 COLO205 SCH 12.36−6.1 4.59 7.54 −5.62 −0.64 −4.31 −0.14 HCT116 BVD −21.09 −5.52 −13.16−13.45 −11.96 −11.15 −13.76 −8.72 HCT116 Vx −11.41 −7.76 −3.07 −13.73−0.59 −18.56 −14.06 −7.79 HCT116 GDC −13.99 −0.25 −1.74 −12.93 −4.85−12.03 −6.32 −4.77 HCT116 SCH −16.35 −7.67 −4.66 −15.32 −12.56 −12.86−10.74 −6.55 HT29 BVD −6.93 −6.46 −1.42 1.49 5.02 17.85 4.74 −2.83 HT29Vx 8.28 −4.28 2.69 5.64 16.94 10.71 4.29 1.36 HT29 GDC −0.88 −3.69 3.56−0.04 10.45 7.56 2.58 3.71 HT29 SCH 7.02 −2.15 2.78 1.25 9.31 13.55 5.7914.05 MIAPaca2 BVD −6.44 −1.23 −15.15 −10.72 −3.27 −8.77 8.62 −7.27MIAPaca2 Vx −0.32 0.61 −1.93 −0.87 −1.58 −5.94 −1.64 −6.58 MIAPaca2 GDC−4.92 −9.44 −8.11 −5.25 −9.8 −7.84 7.4 −1.79 MIAPaca2 SCH 8.35 −9.19−10.88 2.74 1.64 2.11 7.17 −1.88 % change from DMSO (matched cell line)Avg(Bad Avg(ERK 1 Cell Line Treatment S136) Avg(Chk1) Avg(Bim) Avg(AktS473) 2 T202 Y204) A375 BVD 12.31 16.94 14.57 73.07 43.34 A375 Vx 5.06−0.86 12.32 93.85 128.93 A375 GDC −6.21 −7.72 9.8 53.66 142.37 A375 SCH5.32 2.83 17.52 58.13 −90.63 AN3Ca BVD −1.02 −8.45 −12.57 52.11 733.27AN3Ca Vx −3.81 −0.46 −1.46 56.17 718.94 AN3Ca GDC 0.71 9.43 −11.63 82.37645.51 AN3Ca SCH 5.26 1.35 −14.09 66.17 19.75 COLO205 BVD 5.05 −2.8641.73 −5.78 14.39 COLO205 Vx −1.01 −9.16 34.1 −10.96 98.48 COLO205 GDC 45.06 20.59 4.45 20.01 COLO205 SCH 1.88 7.49 29.22 −1.74 −91.43 HCT116BVD −11.49 −11.26 12.44 −6.14 849.12 HCT116 Vx −5.39 −8.63 4.82 −16.94873.33 HCT116 GDC −1.24 2.28 6.2 4.69 526.64 HCT116 SCH −10.55 −5.581.95 −8.76 −75.21 HT29 BVD 10.73 7.4 2.81 −3.06 54.82 HT29 Vx 5.53 4.683.12 −20.5 435.68 HT29 GDC 2 5.68 7.74 −10.5 268.99 HT29 SCH 9.67 14.640.69 −22.14 −74.84 MIAPaca2 BVD −4.12 −3.51 −12.51 9.9 209.14 MIAPaca2Vx 0.36 −1.8 2.1 0.48 729.27 MIAPaca2 GDC 2.25 6.56 5.07 2.24 199.59MIAPaca2 SCH 8.62 12.07 2.08 2.84 −76.71

Example 14 BVD-523 Exhibited Activity in In Vitro Models of BRAF and MEKInhibitor Resistance

Emergence of resistance to BRAF and MEK inhibitors limits their clinicalefficacy. Here, the experiments sought to model and compare thedevelopment of resistance to BRAF (dabrafenib), MEK (trametinib), andERK1/2 (BVD-523) inhibition in vitro. Over several months,BRAF^(V600E)-mutant A375 cells were cultured in progressively increasingconcentrations of each inhibitor. Drug-resistant A375 cell lines werereadily obtained following growth in high concentrations of trametinibor dabrafenib, while developing cell lines with resistance to BVD-523proved challenging (FIG. 34A). Overall, these in vitro data suggest thatat concentrations yielding similar target inhibition, resistance toBVD-523 is delayed compared with dabrafenib or trametinib, and maytranslate to durable responses in the clinic.

Reactivation and dependence on ERK1/2 signaling is a common feature ofacquired resistance to BRAF/MEK inhibition (Morris et al. 2013 andHatzivassiliou et al. 2012); therefore, the activity of BVD-523 in invitro models of acquired resistance was evaluated. First, a dabrafeniband trametinib combination-resistant A375 population was obtained usingthe increased concentration method described. The IC₅₀ and IC₅₀-foldchange from parental A375 for dabrafenib, trametinib, and BVD-523 in theBRAF/MEK combination-resistant population is shown in Table 24. BVD-523IC₅₀ was modestly shifted (2.5-fold), while dabrafenib and trametinibwere more significantly shifted (8.5-fold and 13.5-fold, respectively)(Table 24). The cytotoxic agent paclitaxel was tested as a control withonly a modest shift in potency observed. These data support theinvestigation of BVD-523 in the setting of BRAF/MEK therapy resistance,although the mechanism of resistance in this cell population remains tobe characterized.

TABLE 24 BVD-523 activity in models of BRAF/MEK inhibition Cell LineDabrafenib Trametinib BVD-523 Paclitaxel Parental 2.1 0.2 129 1.9(IC₅₀nM) BRAFi- + 17.9 2.7 323 3.5 MEKi- resistant (IC₅₀nM) Fold +8.5+13.5 +2.5 +1.8 Change

To further investigate the tractability of ERK1/2 inhibition in a modelwith a known mechanism of BRAF inhibitor resistance, AAV-mediated genetargeting was used to generate a pair of RKO BRAF^(V600E)-mutant celllines isogenic for the presence or absence of an engineered heterozygousknock-in of MEK1^(Q56P)-activating mutation (Trunzer et al. 2013 andEmery et al. 2009). MEK1/2 mutations, including MEK1^(Q56P), have beenimplicated in both single-agent BRAF and combination BRAF/MEKtherapy-acquired resistance in patients (Wagle et al. 2011, Wagle et al.2014, Emery et al. 2009 and Johnson et al. 2015). Single-agent assaysdemonstrated that relative to the parental BRAF^(V600E)::MEK1^(wt)cells, the double-mutant BRAF^(V600E)::MEK1^(Q56P) cells displayed amarkedly reduced sensitivity to the BRAF inhibitors vemurafenib anddabrafenib and the MEK inhibitor trametinib (FIG. 34B). In contrast,response to BVD-523 was essentially identical in both the parental andMEK^(Q56P)-mutant cells, indicating that BVD-523 is not susceptible tothis mechanism of acquired resistance. These results were confirmed in 2independently derived double-mutant BRAF^(V600E)::MEK1^(Q56P) cell lineclones, thus validating that results were specifically related to thepresence of the MEK1^(Q56P) mutation rather than an unrelated clonalartifact (data not shown). Similar results were also observed with asecond mechanistically distinct ERK1/2 inhibitor (SCH772984), supportingthe expectation that these observations are specifically related tomechanistic inhibition of ERK1/2 and not due to an off-target compoundeffect.

To further characterize the mechanistic effects of BVD-523 on MAPKpathway signaling in BRAF^(V600E)::MEK1^(Q56P) cell lines, proteinlevels were assessed by Western blot (FIG. 34C). In the parentalBRAF^(V600E) RKO cells, a reduced level of pRSK1/2 was observedfollowing 4-hour treatment with BRAF (vemurafenib), MEK (trametinib), orERK1/2 (BVD-523) inhibitors at pharmacologically active concentrations.In contrast, isogenic double-mutant BRAF^(V600E)::MEK1^(Q56P) cells didnot exhibit reduced RSK phosphorylation following BRAF or MEK inhibitortreatment, while BVD-523 remained effective in inhibiting pRSK1/2 to alevel comparable to parental RKO. Similarly, pRB is reduced, indicatingG0/G1 arrest, by 24 hours of BVD-523 treatment in both parental RKO andBRAF^(V600E)::MEK1^(Q56P).

Acquired KRAS mutations are also known drivers of resistance to MAPKpathway inhibitors. To understand the susceptibility of BVD-523 to thismechanism of resistance, an isogenic panel of clinically relevant KRASmutations in colorectal cell line SW48 was used. Sensitivity to BVD-523was compared with MEK inhibitors selumetinib and trametinib (FIG. 34D).Sensitivity to paclitaxel was unaltered (FIG. 37A). While several mutantKRAS alleles conferred robust to intermediate levels of resistance toMEK inhibition, sensitivity to BVD-523 was unaltered by the majority ofalleles, and where a shift in sensitivity was observed, it was not tothe extent observed with trametinib or selumetinib. Overall, these datasuggest that BVD-523 is more efficacious in this context than MEKinhibitors.

Example 15 BVD-523 Demonstrates In Vivo Activity in a BRAFInhibitor-Resistant Patient-Derived Melanoma Xenograft Model

To confirm and extend the antitumor effects of BVD-523 observed in invitro models of BRAF-/MEK-acquired resistance, a BRAF-resistantxenograft model derived from a patient with resistance to vemurafenibwas utilized. BVD-523 was dosed by oral gavage at 100 mg/kg BID for 28days, both alone and in combination with dabrafenib at 50 mg/kg BID(FIG. 35). As expected, minimal antitumor activity was demonstrated forsingle-agent dabrafenib (22% TGI). BVD-523 activity was significantcompared with vehicle control (P≤0.05), with a TGI of 78%. In thismodel, combining BVD-523 with dabrafenib resulted in a TGI of 76%(P≤0.05); therefore, further benefit was not gained for the combinationcompared with single-agent BVD-523 in this model of BRAF-acquiredresistance.

Example 16 Combination Therapy with BVD-523 and a BRAF InhibitorProvides Promising Antitumor Activity

Patients with BRAF-mutant cancer may acquire resistance to combinedBRAF/MEK therapy (Wagle et al. 2014), warranting consideration of othercombination approaches within the MAPK pathway. The anti-proliferativeeffects of combining BVD-523 with the BRAF inhibitor vemurafenib wasassessed in the BRAF^(V600E)-mutant melanoma cell line G-361. Asanticipated, single agents BVD-523 and vemurafenib were both active, andmodest synergy was observed when combined (FIG. 37B). This indicatesthat BVD-523 combined with BRAF inhibitors are at least additive andpotentially synergistic in melanoma cell lines carrying a BRAF^(V600E)mutation. Furthermore, generating acquired resistance in vitro followingcontinuous culturing of BRAF^(V600E) mutant cell line (A375) in BRAFinhibitor plus BVD-523 was challenging. In contrast generatingresistance to dabrafenib alone occurred relatively rapidly (FIG. 37C).Even resistance to combined dabrafenib and trametinib emerged beforedabrafenib plus trametinib.

The benefit of combined BRAF and ERK inhibition may not be fullyrealized in in vitro combination studies where concentrations are notlimited by tolerability. To understand the benefit of the combination,efficacy was assessed in vivo utilizing xenografts of theBRAF^(V600E)-mutant human melanoma cell line A375. Due to the noteworthyresponse to combination treatment, dosing in the combination groups wasstopped on Day 20 to monitor for tumor regrowth, and was reinitiated onDay 42 (FIG. 36A). Tumors were measured twice weekly until the study wasterminated on Day 45. The median time to endpoint (TTE) for controls was9.2 days, and the maximum possible tumor growth delay (TGD) of 35.8 dayswas defined as 100%. Temozolomide treatment resulted in a TGD of 1.3days (4%) and no regressions. The 50- and 100-mg/kg dabrafenibmonotherapies produced TGDs of 6.9 days (19%) and 19.3 days (54%),respectively, a significant survival benefit (P<0.001), and 1 PR in the100-mg/kg group. The 100-mg/kg BVD-523 monotherapy resulted in a TGD of9.3 days (26%), a significant survival benefit (P<0.001), and 2 durablecomplete responses. The combinations of dabrafenib with BVD-523 eachproduced the maximum possible 100% TGD with noteworthy regressionresponses, and statistically superior overall survival compared withtheir corresponding monotherapies (P<0.001). The lowest dose combinationproduced a noteworthy 7/15 tumor-free survivors (TFS), and the 3higher-dosage combinations produced a total of 43/44 TFS, consistentwith curative or near-curative activity (FIG. 36B). In summary, thecombination of dabrafenib with BVD-523 produced a greater number of TFSand superior efficacy to either single agent.

Based on the activity of BVD-523 plus dabrafenib in A375 xenograftmodels with a starting tumor volume of approximately 75-144 mm³, afollow-up experiment was conducted to determine the efficacy ofcombination therapy in “upstaged” A375 xenografts (average tumor startvolume, 700-800 mm³) (FIG. 36C). The median TTE for controls was 6.2days, establishing a maximum possible TGD of 53.8 days, which wasdefined as 100% TGD for the 60-day study. BVD-523 100-mg/kg monotherapyproduced a negligible TGD (0.7 day, 1%) and no significant survivaldifference from controls (P>0.05). The distribution of TTEs and 2 PRssuggested there may have been a subset of responders to treatment withBVD-523 alone. Dabrafenib 50-mg/kg monotherapy was efficacious, yieldinga TGD of 46.2 days (86%) and a significant survival benefit comparedwith controls (P<0.001). This group had 5 PRs and 5 CRs, including 3TFS, among the 11 evaluable mice (FIG. 36D). Both combinations ofdabrafenib with BVD-523 produced the maximum 100% TGD and a significantsurvival benefit compared with controls (P<0.001). Each combinationproduced 100% regression responses among evaluable mice, though therewere distinctions in regression activity. The 25-mg/kg dabrafenib and50-mg/kg BVD-523 combination had 2 PRs and 8 CRs, with 6/10 TFS, whereasthe 50-mg/kg dabrafenib and 100-mg/kg BVD-523 combination had 11/11 TFSon Day 60 (FIG. 36D). Overall, these data support the rationale forfrontline combination of BVD-523 with BRAF-targeted therapy inBRAF^(V600E)-mutant melanoma, and this is likely to extend to othertumor types harboring this alteration.

Discussion

BVD-523 is a potent, highly selective, reversible, small moleculeATP-competitive inhibitor of ERK1/2 with activity in in vivo and invitro cancer models. In vitro, BVD-523 demonstrated potent inhibitionagainst several human tumor cell lines, particularly those harboringactivating mutations in the MAPK signaling pathway, consistent with itsmechanism of action. BVD-523 elicited changes in downstream target andeffector proteins, including inhibition of direct substrate of ERK1/2,pRSK, and total DUSP6 protein levels. These findings are in line withthose of previous studies of other ERK1/2 inhibitors, which demonstratedeffective suppression of pRSK with ERK1/2 inhibition (Morris et al. 2013and Hatzivassiliou et al. 2012). Interestingly, BVD-523 treatmentresulted in a marked increase in ERK1/2 phosphorylation in vitro and invivo. Similar to our findings, an increase in pERK1/2 has been reportedwith the ERK1/2 inhibitor Vx11e; conversely, pERK1/2 inhibition occurswith SCH772984 (Morris et al. 2013). Although differences in pERK1/2levels were observed among the various ERK1/2 inhibitors tested,downstream effectors (i.e., pRSK1 and total DUSP6) were similarlyinhibited. These findings suggest quantifying ERK1/2 target substrates,such as pRSK1, may serve as reliable pharmacodynamic biomarkers forBVD-523-mediated inhibition of ERK1/2 activity.

While BRAF (dabrafenib, vemurafenib) and MEK (trametinib, cobimetinib)inhibitors validate the MAPK pathway as a therapeutic target,particularly in patients with BRAF^(V600) mutations, the antitumorresponse is limited by the emergence of acquired resistance andsubsequent disease progression. Resistance has been attributed to theupregulation and activation of compensatory signaling molecules(Nazarian et al. 2010, Villanueva et al. 2010, Johannessen et al. 2010and Wang et al. 2011), amplification of the target genes (Corcoran etal. 2010), and activating mutations of pathway components (e.g., RAS,MEK) (Wagle et al. 2011, Emery et al. 2009 and Wang et al. 2011).Reactivation of the ERK1/2 pathway is one common consequence of acquiredresistance mechanism. When introduced into the BRAF^(V600E)-mutantmelanoma cell line A375, MEK^(Q56P) conferred resistance to MEK and BRAFinhibition (Wagle et al. 2011). By contrast, BVD-523 retained its potentinhibitory activity in the engineered MEK^(Q56P) cell line, indicatingthat ERK1/2 inhibition is effective in the setting of upstreamactivating alterations which can arise in response to BRAF/MEKtreatment. As further evidence of a role for BVD-523 in the context ofacquired resistance, efficacy of BVD-523 was evident in a xenograftmodel derived from a tumor sample from a patient whose diseaseprogressed on vemurafenib; the BRAF inhibitor dabrafenib was noteffective in this model. These data support a role for targeting ERK1/2in the setting of BRAF/MEK resistance, and complement previouslypublished findings (Morris et al. 2013 and Hatzivassiliou et al. 2012).To further characterize resistance to inhibitors of the MAPK pathway,the emergence of resistance to BVD-523 itself was investigated. It wasfound that single-agent treatment of cancer cells with BVD-523 wasdurable and more challenging to develop resistance compared with otheragents targeting upstream MAPK signaling components (i.e., dabrafenib,trametinib). This may suggest that acquiring resistance toERK1/2-targeting agents is harder to achieve than acquiring resistanceto BRAF or MEK therapy, potentially due to the fact that BVD-523preferentially targets the more conserved active confirmation of the ATPbinding site. However, in vitro studies with other ERK1/2 inhibitorshave identified specific mutants in ERK1/2 that drive resistance (Jha etal. 2016 and Goetz et al. 2014); these specific mutations have yet to beidentified in clinical samples from ERK1/2 inhibitor-relapsed patients.

The potential clinical benefit of ERK1/2 inhibition with BVD-523 extendsbeyond the setting of BRAF/MEK therapy-resistant patients. As ERK1/2 isa downstream master node within this MAPK pathway, its inhibition isattractive in numerous cancer settings where tumor growth depends onMAPK signaling. Approximately 30% of all cancers harbor RAS mutations;therefore, targeting downstream ERK1/2 with BVD-523 is a rationaltreatment approach for these cancers. Furthermore, results from a studyby Hayes et al. indicate that prolonged ERK1/2 inhibition in KRAS-mutantpancreatic cancer is associated with senescent-like growth suppression(Hayes et al. 2016). However, a combination approach may be required formaximal and durable attenuation of MAPK signaling in the setting of RASmutations. For example, MEK inhibition in KRAS-mutant colorectal cancercell results in an adaptive response of ErbB family activation, whichdampens the response to MEK inhibition (Sun et al. 2014). Similarcontext-specific adaptive responses may occur following ERK1/2inhibition with BVD-523. The optimal treatment combinations for variousgenetic profiles and cancer histologies are the subject of ongoingresearch. In addition to BRAF^(V600) and RAS mutations, otheralterations which drive MAPK are emerging. For example, novel RAFfusions and atypical non-V600 BRAF mutations which promote RAFdimerization activate the MAPK pathway (Yao et al. 2015). BRAFinhibitors such as vemurafenib and dabrafenib which inhibitBRAF^(V600E)-mutant monomer proteins have been shown to be inactive inatypical RAF alterations which drive MAPK signaling in adimerization-dependent manner (Yao et al. 2015). However, treatment withBVD-523 to target downstream ERK1/2 in these tumors may be a novelapproach to addressing this unmet medical need.

In the setting of BRAF^(V600E)-mutant melanoma tumors, combined BRAF andMEK inhibition exemplifies how agents targeting different nodes of thesame pathway can improve treatment response and duration. Ourcombination studies in BRAF^(V600E)-mutant xenografts of human melanomacell line A375 provides support for combination therapy with BVD-523 andBRAF inhibitors. The combination demonstrated superior benefit relativeto single-agent treatments, including results consistent with curativeresponses. The clinical efficacy and tolerability of combinedBRAF/BVD-523 therapy remains to be determined. It would not beunreasonable to expect that a BRAF/ERK1/2 combination will at least becomparable in efficacy to a targeted BRAF/MEK combination. Furthermore,the in vitro observation that acquired resistance to BVD-523 is morechallenging to achieve compared with other MAPK pathway inhibitorssuggests that the BRAF/BVD-523 inhibitor combination has the potentialto provide a more durable response.

Significant progress has also been made using immunotherapy formelanoma. The US FDA has approved various immune checkpoint inhibitorsfor the treatment of advanced melanoma, including the cytotoxicT-lymphocyte antigen-4 targeted agent ipilimumab and the programmeddeath −1 inhibitors pembrolizumab and nivolumab. Combining BVD-523 withsuch immunotherapies is an attractive therapeutic option; furtherinvestigation is warranted to explore dosing schedules and to assesswhether synergistic response can be achieved.

Based on the preclinical data, BVD-523 may hold promise for treatment ofpatients with malignancies dependent on MAPK signaling, including thosewhose tumors have acquired resistance to other treatments. The clinicaldevelopment of BVD-523 is described below. See, Examples 17-24

Example 17 Phase I Dose-Escalation Study of the First-in-Class NovelOral ERK1/2 Kinase Inhibitor BVD-523 (ulixertinib) in Patients WithAdvanced Solid Tumors

The present invention describes the first-in-human dose escalation studyof an ERK1/2 inhibitor for the treatment of patients with advanced solidtumors. BVD-523 has an acceptable safety profile with favorablepharmacokinetics and early evidence of clinical activity.

Mitogen-activated protein kinase (MAPK) signaling via theRAS-RAF-MEK-ERK cascade plays a critical role in oncogenesis; thusattracting significant interest as a therapeutic target. This ubiquitouspathway is composed of RAS upstream of a cascade of the protein kinasesRAF, MEK1/2, and ERK1/2. RAS is activated by GTP binding, which in turnresults in activation of each protein kinase sequentially. Although theyappear to be the only physiologic substrates for MEK1/2, ERK1/2 havemany targets in the cytoplasm and nucleus, including the transcriptionfactors Elk1, c-Fos, p53, Ets1/2, and c-Jun (Shaul et al. 2007). ERK1/2activation and kinase activity influences cellular proliferation,differentiation, and survival through a variety of mechanisms (Rasola etal. 2010), including activation of the ribosomal S6 kinase (RSK) familymembers (Romeo et al. 2012).

Constitutive, aberrant activation of the RAS-RAF-MEK1/2-ERK1/2 signalingpathway has been identified and implicated in the development ormaintenance of many cancers (Schubbert et al. 2007 and Gollob et al.2006). Mutations in RAS family genes, such as KRAS, NRAS, and HRAS arethe most common, with activating RAS mutations occurring in ≈30% ofhuman cancers (Schubbert et al. 2007). KRAS mutations are prevalent inpancreatic (>90%) (Kanda et al. 2012), biliary tract (3%-50%) (Hezel etal. 2014), colorectal (30%-50%) (Arrington et al. 2012), lung (27%)(Pennycuick et al. 2012), ovarian (15%-39%) (Dobrzycka et al. 2009), andendometrioid endometrial (18%) (O'Hara and Bell 2012) cancers; NRASmutations are prevalent in melanoma (20%) (Khattak et al. 2013) andmyeloid leukemia (8%-13%) (Yohe 2015); and HRAS mutations are prevalentin bladder (12%) cancer (Fernandez-Medarde and Santos 2011). Mutationsin RAF family genes, most notably BRAF, are frequent, particularly inmelanoma. BRAF mutations have been identified in 66% of malignantmelanomas and in ˜7% of a wide range of other cancers (Davies et al.2002), while MEK mutations are rarer, occurring at an overall frequencyof 8% in melanomas (Nikolaev et al. 2012). In contrast, ERK mutationsresulting in tumorigenesis have been reported only rarely to date(Deschenes-Simard et al. 2014).

The US Food and Drug Administration (FDA) has approved two selectiveBRAF inhibitors, vemurafenib and dabrafenib, as monotherapies forpatients with BRAF^(V600)-mutant metastatic melanoma (Taflinar [packageinsert] and Zelboraf [package insert]). Though response rates for thesetargeted therapies can be as high as 50% in in patients with BRAF^(V600)mutations, duration of response is often measured in months, not years(Hauschild et al. 2012 and McArthur et al. 2014). The MEK1/2 inhibitortrametinib is also approved as a monotherapy in this setting (Mekinist[package insert]), but is more commonly used in combination with theBRAF inhibitor dabrafenib. First-line use of trametinib administered incombination with dabrafenib offers an even greater improvement inoverall survival compared with vemurafenib monotherapy without increasedoverall toxicity (Robert et al. 2015), highlighting the potentialutility of simultaneously targeting multiple proteins of this MAPKsignaling pathway. This therapeutic combination was also associated witha lower incidence of MEK inhibitor-associated rash and BRAFinhibitor-induced hyperproliferative skin lesions compared with eachsingle agent alone (Flaherty et al. 2012). Recently, a phase III trialalso demonstrated significant improvements in overall survival (25.1 vs.18.7 months, hazard ratio [HR] 0.71, P=0.0107), progression-freesurvival (PFS) (11.0 vs. 8.8 months, HR 0.67, P=0.0004), and overallresponse (69% vs. 53%; P=0.0014) with dabrafenib plus trametinib versusdabrafenib alone in patients with BRAF^(V600E/K) mutation-positivemelanoma (Long et al. 2015). Similarly, significant improvements in PFS(9.9 vs. 6.2 months, HR 0.51, P<0.001) and the rate of complete response(CR) or partial response (PR) (68% vs. 45%; P<0.001) have beendemonstrated with the combination of cobimetinib plus vemurafenibcompared with vemurafenib alone (Larkin et al. 2014). To this end, FDAapproval was recently granted for the combination of vemurafenib andcobemetinib for BRAF^(V600E/K)-mutated melanoma. Based on these andrelated findings, the combination of a BRAF inhibitor plus a MEKinhibitor has become a standard targeted treatment option for patientswith metastatic melanoma containing BRAF^(V600E/K) mutations.

Though BRAF/MEK-targeted combination therapy has been demonstrated toprovide significant additional benefit beyond single-agent options, mostpatients eventually develop resistance and disease progression after ˜12months (Robert et al. 2015, Flaherty et al. 2012 and Long et al. 2015).Several mechanisms of acquired resistance following either single-agentor combination therapies have been identified, including the generationof BRAF splicing variants, BRAF amplification, development of NRAS orMEK mutations, and upregulation of bypass pathways (Poulikakos et al.2011, Corcoran et al. 2010, Nazarian et al. 2010, Shi et al. 2014,Johannessen et al. 2010, Wagle et al. 2011, Wagle et al. 2014 andAhronian et al. 2015). Central to many of these mechanisms of resistanceis the reactivation of ERK signaling, which enables the rapid recoveryof MAPK pathway signaling and escape of tumor cells from single-agentBRAF or combination BRAF/MEK inhibitor therapies (Paraiso et al. 2010).ERK inhibition may provide the opportunity to avoid or overcomeresistance from upstream mechanisms, as it is the most distal masterkinase of this MAPK signaling pathway. This is supported by preclinicalevidence that inhibition of ERK by small molecule inhibitors acted toboth inhibit the emergence of resistance and overcome acquiredresistance to BRAF and MEK inhibitors (Morris et al. 2013 andHatzivassiliou et al. 2012).

BVD-523 is a highly potent, selective, reversible, ATP-competitiveERK1/2 inhibitor which has been shown to reduce tumor growth and inducetumor regression in BRAF and RAS mutant xenograft models. Furthermore,single-agent BVD-523 inhibited human xenograft models that werecross-resistant to both BRAF and MEK inhibitors. See, Examples 9-16.Therefore, an open-label, first-in-human study (Clinicaltrials.govidentifier, NCT01781429) of oral BVD-523 to identify both the maximumtolerated dose and the recommended dose for further study wasundertaken. The present study also aimed to assess pharmacokinetic andpharmacodynamic properties as well as preliminary efficacy in patientswith advanced cancers.

Example 18 Patient Characteristics

A total of 27 patients were enrolled and received at least one dose ofstudy drug from Apr. 4, 2013 to Dec. 1, 2015. Baseline demographics anddisease characteristics are shown in Table 25. The median patient agewas 61 years (range, 33-86 years). Fifty-two percent (14/27) of patientswere male and 63% (17/27) had an Eastern Cooperative Oncology Group(ECOG) performance status of 1. Melanoma was the most common cancer(30%; BRAF mutation present in 7/8 of these patients). The remainingpatients had colorectal (19%; 5/27), papillary thyroid (15%; 4/27), ornon-small cell lung cancer (NSCLC) (7%; 2/27), and 8 (30%) wereclassified as having other cancers (2 pancreatic, 1 appendiceal, 1nonseminomatous germ cell, 1 ovarian and 3 with unknown primary). Themajority of patients had received 2 or more prior lines of systemictherapy, with 41% (11/27) receiving 2 to 3 and 48% (13/27) receiving >3prior lines of systemic therapy.

TABLE 25 Baseline demographics and clinical characteristics of patientsParameter N = 27 Median age, years (range) 61 (33-86) Sex, n (%) Female13 (48) Male 14 (52) Ethnicity, n (%) Not Hispanic/Latino 27 (100) ECOGperformance status  0 10 (37)  1 17 (63) Cancer type, n (%) Melanoma^(a)8 (30) Colorectal 5 (19) Papillary thyroid 4 (15) Non-small cell lung 2(7) Other^(b) 8 (30) Molecular abnormalities, n (%)^(c) BRAF mutant 13(48) KRAS mutant 6 (22) NRAS mutant 2 (7) Other^(d) 7 (26) Unknown 4(15) Number of prior systemic anticancer regimens, n (%)  0 1 (4)  1 2(7) 2-3 11 (41) >3 13 (48) Prior BRAF/MEK-targeted therapy^(e), n (%) 11(41) BRAF 5 (19) MEK 6 (22) BRAF/MEK 2 (7) ^(a)Seven were BRAF mutantand 1 was unknown. ^(b)Two pancreatic, 1 appendiceal, 1 non-seminomatousgerm cell, 1 ovarian, 3 unknown primary. ^(c)Patients may have more than1 molecular abnormality. ^(d)Other molecular abnormalities includedERCC1, RRM1, thymidylate synthetase, GNAS, MEK1, TP53, CREBBP, ROS1,PTEN, AKT3, and PIK3CA. ^(e)Some patients were treated with more thanone BRAF inhibitor. Abbreviation: ECOG, Eastern Cooperative OncologyGroup.

Example 19 Ex Vivo Effects of BVD-523 on RSK1/2 Phosphorylation

An ex vivo biomarker assay that could be used to support clinicalstudies was developed to demonstrate the inhibitory effects of BVD-523on ERK activity. The assay extends preclinical cellular data whereinhibitors of MAPK signaling, such as BVD-523, dabrafenib, trametinib,and vemurafenib, have been shown to inhibit RSK phosphorylation as afunction of inhibitor concentration in BRAF mutant cancer cell lines.See, Examples 9-16. Specifically, ERK inhibitor-dependent inhibition ofphorbol 12-myristate 13-acetate (PMA)-stimulated phosphorylation of theERK substrate RSK1 in whole blood was used as a target marker. WhenBVD-523 was added directly to whole blood from healthy volunteers,PMA-stimulated RSK phosphorylation decreased with increasingconcentrations of BVD-523 (FIG. 38). The mean IC₅₀ for the cumulativedata was 461±20 nM for BVD-523, with a maximum inhibition of 75.8±2.7%at 10 μM BVD-523. Maximum inhibition was defined as the RSKphosphorylation measured in the presence of 10 μM BVD-523.Patient-derived whole blood samples, collected just prior to dosing orat defined timepoints following dosing with BVD-523, were similarlytreated and RSK phosphorylation levels quantitated.

Example 20 Dose Escalation, Dose-Limiting Toxicities (DLTs), MaximumTolerated Dose (MTD), and Recommended Phase II Dose (RP2D)

As per protocol, 5 single-patient cohorts (from 10 to 150 mg twice-daily[BID]) proceeded without evidence of a DLT. The 300-mg BID cohort wasexpanded to more fully characterize BVD-523 exposures. One of 6 patientsgiven 600 mg BID experienced a DLT of Grade 3 rash. The 900-mg BID doseexceeded the MTD, with one patient experiencing Grade 3 pruritus andelevated aspartate aminotransferase (AST) and another patientexperiencing Grade 3 diarrhea, vomiting, dehydration, and elevatedcreatinine (Table 26). The subsequent intermediate dose of 750 mg BIDalso exceeded the MTD, with DLTs of Grade 3 rash and Grade 2 diarrhea in1 patient and Grade 2 hypotension, elevated creatinine, and anemia inanother patient. Therefore, the MTD and RP2D were determined to be 600mg BID.

TABLE 26 Dose-limiting toxicities in Cycle 1 (21 days) Dose, mg DLT(BID) Frequency DLT Description 10 0/1 N/A 20 0/1 N/A 40 0/1 N/A 75 0/1N/A 150  0/1 N/A 300  0/4 N/A 600  1/8 Rash (Grade 3) 750^(a ) 2/4 Rash(Grade 3), diarrhea (Grade 2) Hypotension (Grade 2), elevated creatinine(Grade 2), anemia (Grade 2), delay to cycle 2 dosing 900  2/7 Pruritus(Grade 3), elevated AST (Grade 3) Diarrhea (Grade 3), vomiting (Grade3), dehydration (Grade 3), elevated creatinine (Grade 3)^(a)Intermediate dose. Abbreviations: AST, aspartate transaminase, BID,twice daily; DLT, dose-limiting toxicity; N/A, not applicable.

Example 21 Adverse Events (AEs)

Investigator-assessed treatment-related AEs of any grade were noted in26 of 27 patients (96%). The most common treatment-related AEs (>30%)were rash (predominately acneiform) (70%), fatigue (59%), diarrhea(52%), and nausea (52%) (Table 27). No patients experienced a Grade 4 or5 treatment-related AE or discontinued treatment due to atreatment-related AE. Most events were Grade 1 to 2, withtreatment-related Grade 3 events noted in 13 of 27 patients (48%). Theonly Grade 3 treatment-related events present in ≥10% of patients werediarrhea (15%) and increased liver function tests (11%), all of whichoccurred above the 600-mg BID dose.

TABLE 27 Adverse events possibly/definitely related to BVD-523 in ≥10%of patients N = 27 Any grade, n Grade 1 or 2, Grade 3^(a), n Event (%) n(%) (%) Rash 20 (74)  18 (67)  2^(b) (7)  Fatigue 17 (63)  16 (59)  1(4) Diarrhea 16 (59)  12 (44)   4 (15) Nausea 14 (52)  14 (52)  0Vomiting 8 (30) 7 (26) 1 (4) Anorexia 6 (22) 6 (22) 0 Pruritus 6 (22) 6(22) 0 Anemia 5 (19) 3 (11) 2 (7) Increased creatinine 5 (19) 4 (15) 1(4) Dehydration 5 (19) 3 (11) 2 (7) Peripheral edema 4 (15) 4 (15) 0Increased LFTs (ALT 4 (14) 1 (4)   3 (11) and AST) Blurry/dimmedvision^(c) 3 (11) 3 (11) 0 Constipation 3 (11) 3 (11) 0 Fever 3 (11) 3(11) 0 ^(a)No patients experienced Grade 4 or 5 AEs that were possiblyor definitely related to BVD-523 treatment. ^(b)Acneiform andmaculo-papular rash. ^(c)One Grade 1 event of related central serousretinopathy. Analysis cut-off date: Dec. 1, 2015. Abbreviations: AEs,adverse events; ALT, alanine transaminase; AST, aspartate transaminase;LFTs, liver function tests.

Fourteen patients experienced a total of 28 serious AEs (SAEs). Nine ofthese were considered to be related or possibly related to BVD-523 bythe investigator, which included dehydration, diarrhea, or elevatedcreatinine (2 patients each), vomiting, nausea, and fever (1 patienteach). All other SAEs were considered to be unrelated to treatment withBVD-523. Dose reductions resulting from AEs occurred in 3 patientsduring the study: 1 patient reduced from 600 mg BID to 300 mg BID and 2patients reduced from 900 mg BID to 600 mg BID.

Example 22 Pharmacokinetics

Single-dose and steady-state pharmacokinetics of BVD-523 are summarizedin FIG. 39A and Table 28. Generally, orally administered BVD-523 wasslowly absorbed in patients with advanced malignancies. After reachingthe maximum concentration (C_(max)), plasma BVD-523 levels remainedsustained for approximately 2 to 4 hours. Subsequently, plasma drugconcentrations slowly declined. Since plasma drug concentrations weremeasured only up to 12 hours after the morning dose, it was not possibleto calculate an effective or terminal phase elimination rate. BVD-523pharmacokinetics were linear and dose proportional in terms of both C.and area under the curve (AUC) when administered up to 600 mg BID. Afurther increase in exposure was not observed as the dose increased from600 to 900 mg BID. The C_(max) reached the level of the EC₅₀ based onthe ex vivo whole blood assay (200 ng/mL) for all doses above 20 mg BID.Additionally, steady-state exposures remained at or above the targetEC₅₀ for dose levels of 150 mg BID throughout the dosing period. Minimalplasma accumulation of BVD-523 and its metabolites were observed on Day15 at the lower (<75 mg BID) dose levels, whereas accumulation rangedfrom approximately 1.3- to 4.0-fold for the higher dose levels. Predoseconcentrations on Day 22 were generally similar to those on Day 15,indicating that steady state had already been attained by Day 15 (datanot shown). The degree of interpatient variability in plasma exposure toBVD-523 and its metabolites was considered moderate and not problematic.

TABLE 28 Steady-state BVD-523 pharmacokinetics (Cycle 1, Day 15) Dose,C_(max), ng/mL ± SD AUC_(0.22), ng · hr/mL ± SD mg^(a) n∞ Day 1 Day 15Day 1 Day 15  10 1 48.2 45.7 220 234  20 1 14.9 15.8 91.7 98.7  40 1 100191 614 999 150 1 133 326 817 2770 300 4^(b) 765 ± 234 586 ± 257   4110± 1140 4460 ± 2460  600^(c) 7^(d) 1110 ± 589  2750 ± 1740   2750 ± 174024400 ± 16200 750 4^(b) 1450 ± 539  2290 ± 1790^(f) 10700 ± 1120^(g) 23300 ± 19800^(f) 900 7^(e) 1430 ± 1010 1720 ± 328   10800 ± 6320^(h)15900 ± 1300^(g)   ^(a)Dose level administered twice daily; ^(b)N = 3 onDay 15; ^(c)Number of subjects for Day 15 at the 600 mg dose levelincludes two subjects who started Day 1 dosing at 900 mg and were laterreduced to 600 mg; ^(d)n = 8 on Day 15; ^(e)n = 4 on Day 15; ^(f)Onesubject started on Day 1 dosing at 750 mg and was later reduced to 450mg. Day 15 parameters for this subject reflect at least 10 consecutivedoses at 450 mg/dose. Individual Day 15 parameters were 1300 ng/ml, forC_(max) and 10700 ng · hr/mL for AU₀₋₂₂; ^(g)n = 3; ^(h)n = 5.

The urinary excretion after first dose and at steady state of BVD-523was negligible (<0.2% of the dose) at all dose levels within 12 hourspostdose, and not dose-related within this very low percentage range.Renal clearance appeared to be dose-independent. Individual renalclearance values ranged from 0.128 to 0.0895 L/hr (where n=1 per doselevel) and mean values ranged from 0.0149 to 0.0300 L/hr (where n 3).

Example 23 Pharmacodynamic Confirmation of Target Inhibition by BVD-523

To confirm on-target and pathway inhibition by BVD-523, RSK-1phosphorylation was examined as a target biomarker in human whole bloodsamples from patients with solid tumors who received BVD-523. Steadystate whole blood samples collected just prior to Day 15 dosing fromBVD-523-treated patients displayed concentration-dependent inhibition ofPMA stimulated ERK activity (FIG. 39B), ranging from 0% ERK inhibitionwith BVD-523 dosing at 10 mg BID to 93±8% ERK inhibition with dosing at900 mg BID. The plasma concentrations of BVD-523 that yielded 50%inhibition of ERK phosphorylation were similar whether BVD-523 wasspiked directly into healthy volunteer plasma or was present followingoral dosing of patients.

Example 24 Antitumor Effects

Tumor response to BVD-523 was assessed in 25 evaluable patients usingResponse Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1);2 patients did not receive both scans of target lesions and were thusnot evaluated using RECIST v1.1. No patients achieved a completeresponse, but 3 patients (all patients with melanoma with BRAF^(V600)mutations) achieved a partial response (129 days [BRAF/MEK-inhibitornaïve], 294 days ongoing at [refractory to prior BRAF/MEK inhibitors],313 days ongoing by the data cutoff date [intolerant to other BRAF/MEKinhibitors]) (FIG. 40A). Interestingly, all 3 partial responders hadBRAF-mutant melanoma. One partial responder, who was receiving BVD-523at a dose of 450 mg BID, had an approximate 70% reduction in the sum oftarget lesions from baseline, while the other partial responders showedreductions of 47.0% and 33.6%. Stable disease was demonstrated in 18patients, with 6 having stable disease for more than 6 months, and 6additional patients having stable disease for more than 3 months. Inthis study, 4 patients displayed progressive disease at firstevaluation.

FIG. 40B shows computed tomography (CT) scans of 1 of the 3 partialresponders (RECIST v1.1) who had progressed on prior vemurafenib andsubsequent dabrafenib/trametinib treatment; a durable partial responsewas observed following dosing of BVD-523 600 mg BID for >300 days.BVD-523 was associated with a metabolic response usingfluorodeoxyglucose-positive emission tomography (¹⁸F-FDG-PET) in 5 of 16evaluable patients.

FIG. 41 depicts the time to response and the duration of response in thestudy population. The two patients who demonstrated responses to BVD-523remained on study and continued with BVD-523 treatment as of the studycutoff date (>500 days); additionally, one patient with bronchoalveolarNSCLC (not enough tissue for molecular profiling) had been on treatmentfor >700 days with stable disease. Twenty-four of 27 patients (90%)discontinued treatment due to progressive disease (22/27, 82%) or otherreasons (2/27, 7%). The mean duration of BVD-523 treatment beforediscontinuation was 4.7 months.

Discussion

The present invention presents results from a first-in-human studyevaluating the safety, pharmacokinetics, pharmacodynamics, andpreliminary efficacy of BVD-523 in 27 patients with advanced solidtumors. In this dose-escalation study, oral treatment with BVD-523resulted in both radiographic responses by RECIST v1.1 (3 partialresponses) and prolonged disease stabilization in some patients, themajority of whom had been treated with prior systemic therapies.Evidence of BVD-523-dependent inhibition of metabolic response in tumorswas established in a subset of patients by imaging tumor uptake of¹⁸F-glucose. Drug exposures increased linearly with increasing doses upto 600 mg BID, with exposures at 600 mg BID providing near complete 24/7inhibition of ERK-dependent substrate (RSK-1) phosphorylation in an exvivo whole blood assay. Furthermore, tolerability to BVD-523 wasmanageable when administered up to its MTD and RP2D, determined to be600 mg BID.

BVD-523 was generally well tolerated, with manageable and reversibletoxicity. The most common AEs were rash (usually acneiform), fatigue,and gastrointestinal side effects, including nausea, vomiting, anddiarrhea. The safety profile of BVD-523 is consistent with its selectiveinhibition of the MAPK pathway; the AE profile shows considerableoverlap with MEK inhibitor experience. However, toxicities associatedwith any targeted therapy may include dependence on both the specificmechanism and the degree of target inhibition as well as any off-targeteffects (Zelboraf [package insert] and Hauschild et al. 2012). Ongoingand future investigations will extend both the efficacy and safetyprofile demonstrated in this dose-escalation study, and will guide howthe unique profile of the ERK inhibitor BVD-523 might be used as asingle agent or in combination with other agents.

Durable responses by RAF and MEK inhibitors are often limited byintrinsic and eventual acquired resistance, with a common feature ofteninvolving reactivation of the ERK pathway (Poulikakos et al. 2011,Corcoran et al. 2010, Nazarian et al. 2010, Shi et al. 2014, Johannessenet al. 2010, Wagle et al. 2011, Wagle et al. 2014, Ahronian et al. 2015and Paraiso et al. 2010). Thus, ERK inhibition with BVD-523 alone or incombination with other MAPK signaling pathway inhibitors may have thepotential to delay the development of resistance to existing therapiesand to benefit a broader patient population. That ERK inhibitors,including BVD-523, retain their potency in BRAF- and MEK-resistant celllines provide preclinical evidence for the use of ERK inhibitors inpatients with acquired resistance to standard of care (BRAF/MEKcombination therapy) See, e.g., Examples 9-16. Importantly, in thisstudy, a patient whose cancer had progressed after experiencing stabledisease when treated initially with a BRAF inhibitor (vemurafenib) andsubsequently with a combination of BRAF and MEK inhibitors(dabrafenib/trametinib) had a partial response when receivingsingle-agent BVD-523. This patient has remained on-study for a total of708 days, as of the cutoff date of the study reported herein. Based inpart on the antitumor effects observed in this patient, the FDA hasdesignated as a Fast Track development program the investigation ofBVD-523 for the treatment of patients with unresectable or metastaticBRAF^(v600) mutation-positive melanoma that is refractory to or hasprogressed following treatment with a BRAF and/or MEK inhibitor(s).Precise definition of exactly how BVD-523 might best support patientcare (eg, as a single agent or in various combinations) requiresadditional clinical studies.

In summary, the present examples present data from an initial data fromthe dose escalation portion of a phase I study evaluating BVD-523, anovel first-in-class ERK inhibitor, as a treatment for patients withadvanced cancers. Continuous, twice-daily oral treatment with BVD-523resulted in antitumor effects in several patients, including patientseither naïve to or having progressed on available MAPK pathway-targetedtherapies. BVD-523 was generally well tolerated in this advanced cancerpatient population and toxicities were manageable; the MTD and RP2D were600 mg BID. BVD-523 exposures increased linearly up to the RP2D androbust pharmacodynamics effects were evident at this dose level. Anexpansion of this phase I clinical study is currently underway toconfirm and extend the observations made in the dose-escalation phase.Specifically, patients are being enrolled into molecularly classifiedexpansion cohorts (e.g., NRAS, BRAF, MEK or ERK alterations) acrossvarious tumor histologies. Furthermore, expansion cohorts are evaluatingthe use of BVD-523 in patients with cancer who are either naïve toavailable MAPK pathway therapies or those whose disease has progressedon such treatments.

DOCUMENTS

-   ABSALAN, Farnaz, Mostafa Ronaghi (2008). Molecular Inversion Probe    Assay. Methods in Molecular Biology 396. Humana Press. pp. 315-330.-   AHRONIAN L G, Sennott E M, Van Allen E M, Wagle N, Kwak E L, Faris J    E, et al. Clinical acquired resistance to RAF inhibitor combinations    in BRAF-mutant colorectal cancer through MAPK pathway alterations.    Cancer Discov 2015; 5:358-67.-   ARCILA M E, Drilon A, Sylvester B E, Lovly C M, Borsu L, Reva B, et    al. MAP2K1 (MEK1) mutations define a distinct subset of lung    adenocarcinoma associated with smoking. Clin Cancer Res 2015;    21:1935-43.-   ARONOV A M, Baker C, Bemis G W, Cao J, Chen G, Ford P J, et al.    Flipped out: structure-guided design of selective pyrazolylpyrrole    ERK inhibitors. J Med Chem 2007; 50:1280-7.-   ARONOV A M, Tang Q, Martinez-Botella G, Bemis G W, Cao J, Chen G, et    al. Structure-guided design of potent and selective pyrimidylpyrrole    inhibitors of extracellular signal-regulated kinase (ERK) using    conformational control. J Med Chem 2009; 52:6362-8.-   ARRINGTON A K, Heinrich E L, Lee W, Duldulao M, Patel S, Sanchez J,    et al. Prognostic and predictive roles of KRAS mutation in    colorectal cancer. Int J Mol Sci 2012; 13:12153-68.-   CARGNELLO M, Roux P P. Activation and function of the MAPKs and    their substrates, the MAPK-activated protein kinases. Microbiol Mol    Biol Rev 2011; 75:50-83.-   CARLINO M S, Fung C, Shahheydari H, Todd J R, Boyd S C, Irvine M, et    al. Preexisting MEK1P124 mutations diminish response to BRAF    inhibitors in metastatic melanoma patients. Clin Cancer Res 2015;    21:98-105.-   CHAPMAN P B, Hauschild A, Robert C, Haanen J B, Ascierto P, Larkin    J, et al. Improved survival with vemurafenib in melanoma with BRAF    V600E mutation. N Engl J Med 2011; 364:2507-16.-   CORCORAN, R. B., et al. BRAF gene amplification can promote acquired    resistance to MEK inhibitors in cancer cells harboring the BRAF    V600E mutation. Sci Signal (2010); 3(149): ra84.-   DAI, B., et al. STAT3 mediates resistance to MEK inhibitor through    microRNA miR-17. Cancer Res (2011); 71:3658-3668.-   DAVIES H, Bignell G R, Cox C, Stephens P, Edkins S, Clegg S, et al.    Mutations of the BRAF gene in human cancer. Nature 2002; 417:949-54.-   DESCHENES-SIMARD X, Kottakis F, Meloche S, Ferbeyre G. ERKs in    cancer: friends or foes? Cancer Res 2014; 74:412-9.-   DOBRZYCKA B, Terlikowski S J, Kowalczuk O, Niklinska W, Chyczewski    L, Kulikowski M. Mutations in the KRAS gene in ovarian tumors. Folia    Histochem Cytobiol 2009; 47:221-4.-   EMERY, C. M., et al. MEK1 mutations confer resistance to MEK and    B-RAF inhibition. PNAS (2009); 106(48):20411-6.-   FEDOROV O, Niesen F H, Knapp S. Kinase inhibitor selectivity    profiling using differential scanning fluorimetry. Methods Mol Biol    2012; 795:109-18.-   FERNÁNDEZ-MEDARDE A, Santos E. Ras in cancer and developmental    diseases. Genes Cancer 2011; 2:344-58.-   FLAHERTY K T, Infante J R, Daud A, Gonzalez R, Kefford R F, Sosman    J, et al. Combined BRAF and MEK inhibition in melanoma with BRAF    V600 mutations. N Engl J Med 2012; 367:1694-703.-   GOETZ E M, Ghandi M, Treacy D J, Wagle N, Garraway L A. ERK    mutations confer resistance to mitogen-activated protein kinase    pathway inhibitors. Cancer Res 2014; 74:7079-89.-   GOLLOB J A, Wilhelm S, Carter C, Kelley S L. Role of Raf kinase in    cancer: therapeutic potential of targeting the Raf/MEK/ERK signal    transduction pathway. Semin Oncol 2006; 33:392-406.-   GREGER, James G., et al. “Combinations of BRAF, MEK, and PI3K/mTOR    inhibitors overcome acquired resistance to the BRAF inhibitor    GSK2118436 dabrafenib, mediated by NRAS or MEK mutations.” Molecular    cancer therapeutics 11.4 (2012): 909-920.-   GROENENDIJK F H, Bernards R. Drug resistance to targeted therapies:    deja vu all over again. Mol Oncol 2014; 8:1067-83.-   HALL R D, Kudchadkar R R. BRAF mutations: signaling, epidemiology,    and clinical experience in multiple malignancies. Cancer Control    2014; 21:221-30.-   HARDENBOL, P., et al. Multiplexed genotyping with sequence-tagged    molecular inversion probes. Nat. Biotechnol. 2003, no. 21, p.    673-678.-   HATZIVASSILIOU, Georgia, et al. “RAF inhibitors prime wild-type RAF    to activate the MAPK pathway and enhance growth.” Nature 464.7287    (2010): 431-435.-   HATZIVASSILIOU G, Liu B, O'Brien C, Spoerke J M, Hoeflich K P,    Haverty P M, et al. ERK inhibition overcomes acquired resistance to    MEK inhibitors. Mol Cancer Ther 2012; 11:1143-54.-   HAUSCHILD A, Grob J-J, Demidov L V, Jouary T, Gutzmer R, Millward M,    et al. Dabrafenib in BRAF-mutated metastatic melanoma: a    multicentre, open-label, phase 3 randomised controlled trial. Lancet    2012; 380:358-65.-   HAYES T K, Neel N F, Hu C, Gautam P, Chenard M, Long B, et al.    Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is    Associated with MYC Degradation and Senescence-like Growth    Suppression. Cancer Cell 2016; 29:75-89.-   HEZEL A F, Noel M S, Allen J N, Abrams T A, Yurgelun M, Faris J E,    et al. Phase II study of gemcitabine, oxaliplatin in combination    with panitumumab in KRAS wild-type unresectable or metastatic    biliary tract and gallbladder cancer. Br J Cancer 2014; 111:430-6.-   JHA S, Morris E J, Hruza A, Mansueto M S, Schroeder G, Arbanas J, et    al. Dissecting therapeutic resistance to ERK inhibition. Mol Cancer    Ther 2016; 15:548-59.-   JOHANNESSEN, C. M., et al. COT/MAP3K8 drives resistance to RAF    inhibition through MAP kinase pathway reactivation. Nature (2010);    468(7326):968-972.-   JOHNSON D B, Menzies A M, Zimmer L, Eroglu Z, Ye F, Zhao S, et al.    Acquired BRAF inhibitor resistance: A multicenter meta-analysis of    the spectrum and frequencies, clinical behaviour, and phenotypic    associations of resistance mechanisms. Eur J Cancer 2015; 51:2792-9.-   KANDA M, Matthaei H, Wu J, Hong S M, Yu J, Borges M, et al. Presence    of somatic Mutations in most early-stage pancreatic intraepithelial    neoplasia. Gastroenterology 2012; 142:730-733.-   KHATTAK M, Fisher R, Turajlic S, Larkin J. Targeted therapy and    immunotherapy in advanced melanoma: an evolving paradigm. Ther Adv    Med Oncol 2013; 5:105-18.-   KING, Alastair J., et al. “Dabrafenib; preclinical characterization,    increased efficacy when combined with trametinib, while BRAF/MEK    tool combination reduced skin lesions.” PloS one 8.7 (2013): e67583.-   LARKIN J, Ascierto P A, Dreno B, Atkinson V, Liszkay G, Maio M, et    al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N    Engl J Med 2014; 371:1867-76.-   LITTLE, A. S., et al., Amplification of the Driving Oncogene, KRAS    or BRAF, Underpins Acquired Resistance to MEK1/2 Inhibitors in    Colorectal Cancer Cells. Sci. Signal. 4, ra17 (2011).-   LIU, Dingxie, et al. “BRAF V600E maintains proliferation,    transformation, and tumorigenicity of BRAF-mutant papillary thyroid    cancer cells.” Journal of Clinical Endocrinology & Metabolism 92.6    (2007): 2264-2271.-   LIU B, Fu L, Zhang C, Zhang L, Zhang Y, Ouyang L, et al.    Computational design, chemical synthesis, and biological evaluation    of a novel ERK inhibitor (BL-EI001) with apoptosis-inducing    mechanisms in breast cancer. Oncotarget 2015; 6:6762-75.-   LONG G V, Fung C, Menzies A M, Pupo G M, Carlino M S, Hyman J, et    al. Increased MAPK reactivation in early resistance to    dabrafenib/trametinib combination therapy of BRAF-mutant metastatic    melanoma. Nat Commun 2014; 5:5694.-   LONG G V, Stroyakovskiy D, Gogas H, Levchenko E, de Braud F, Larkin    J, et al. Dabrafenib and trametinib versus dabrafenib and placebo    for Val600 BRAF-mutant melanoma: a multicentre, double-blind, phase    3 randomised controlled trial. Lancet 2015; 386:444-51.-   MANANDHAR S P, Hildebrandt E R, Schmidt W K. Small-molecule    inhibitors of the Rce1p CaaX protease. J Biomol Screen. 2007;    12(7):983-993.-   MASSEY P R, Prasad V, Figg W D, Fojo T. Multiplying therapies and    reducing toxicity in metastatic melanoma. Cancer Biol Ther 2015;    16:1014-8.-   MAURER, T, Garrenton, L S, Oh, A, Pitts, K, Anderson, D J, Skelton,    N J, Fauber, B P, Pan, B, Malek, S, Stokoe, D, Ludlam, M J C,    Bowman, K K, Wu, J, Giannetti, A M, Starovasnik, M A, Mailman, I,    Jackson, P K, Rudolph, J, Wang, W, Fang, G. Small-molecule ligands    bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide    exchange activity. PNAS. 2012; 109(14):5299-304.-   MCARTHUR G A, Chapman P B, Robert C, Larkin J, Haanen J B, Dummer R,    et al. Safety and efficacy of vemurafenib in BRAF^(V600E) and    BRAF^(v600K) mutation-positive melanoma (BRIM-3): extended follow-up    of a phase 3, randomised, open-label study. Lancet Oncol 2014;    15:323-32.-   MEKINIST [package insert]. Research Triangle Park, N C:    GlaxoSmithKline; 2014.-   METZKER, Emerging technologies in DNA sequencing Genome Res. 2005.    15: 1767-1776.-   MITTAL, Rohit et al. “The acetyltransferase activity of the    bacterial toxin YopJ of Yersinia is activated by eukaryotic host    cell inositol hexakisphosphate.” Journal of Biological Chemistry    285.26 (2010): 19927-19934.-   MORRIS E J, Jha S, Restaino C R, Dayananth P, Zhu H, Cooper A, et    al. Discovery of a novel ERK inhibitor with activity in models of    acquired resistance to BRAF and MEK inhibitors. Cancer Discov 2013;    3:742-50.-   NAZARIAN, R., et al. Melanomas acquire resistance to B-RAF (V600E)    inhibition by RTK or N-RAS upregulation. Nature. 2010;    468(7326):973-977.-   NIKOLAEV S I, Rimoldi D, Iseli C, Valsesia A, Robyr D, Gehrig C, et    al. Exome sequencing identifies recurrent somatic MAP2K1 and MAP2K2    mutations in melanoma. Nat Genet 2012; 44:133-9.-   NILSSON, M., et al. Padlock probes: circularizing oligonucleotides    for localized DNA detection. Science. 1994, no. 265, p. 2085-2088.-   O′HARA A J, Bell D W. The genomics and genetics of endometrial    cancer. Adv Genomics Genet 2012; 2012:33-47.-   OJESINA A I, Lichtenstein L, Freeman S S, Pedamallu C S,    Imaz-Rosshandler I, Pugh T J, et al. Landscape of genomic    alterations in cervical carcinomas. Nature 2014; 506:371-5.-   OTA et al., Single nucleotide polymorphism detection by polymerase    chain reaction-restriction fragment length polymorphism. Nat Protoc.    2007; 2(11):2857-64.-   PARAISO KHT, Fedorenko I V, Cantini L P, Munko A C, Hall M, Sondak V    K, et al. Recovery of phospho-ERK activity allows melanoma cells to    escape from BRAF inhibitor therapy. Br J Cancer 2010; 102:1724-30.-   PATGIRI A, Yadav, K K, Arora, P S, Bar-Sagi, D. An orthosteric    inhibitor of the Ras-Sos interaction. Nat Chem Biol. 2011;    7:585-587.-   PENNYCUICK A, Simpson T, Crawley D, Lal R, Santis G, Cane P, et al.    Routine EGFR and KRAS mutation analysis using COLD-PCR in non-small    cell lung cancer. Int J Clin Pract 2012; 66:748-52.-   PORTER S B, Hildebrandt E R, Breevoort S R, Mokry D Z, Dore™,    Schmidt W K. Inhibition of the CaaX proteases Rce1p and Ste24p by    peptidyl (acyloxy)methyl ketones. Biochim Biophys Acta. 2007;    1773(6):853-862.-   POULIKAKOS P I, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G,    et al. RAF inhibitor resistance is mediated by dimerization of    aberrantly spliced BRAF(V600E). Nature 2011; 480:387-90.-   QUEIROLO P, Picasso V, Spagnolo F. Combined BRAF and MEK inhibition    for the treatment of BRAF-mutated metastatic melanoma. Cancer Treat    Rev 2015; 41:519-26.-   RASOLA A, Sciacovelli M, Chiara F, Pantic B, Brusilow W S,    Bernardi P. Activation of mitochondrial ERK protects cancer cells    from death through inhibition of the permeability transition. Proc    Natl Acad Sci USA 2010; 107:726-31.-   RIZOS H, Menzies A M, Pupo G M, Carlino M S, Fung C, Hyman J, et al.    BRAF inhibitor resistance mechanisms in metastatic melanoma:    spectrum and clinical impact. Clin Cancer Res 2014; 20:1965-77.-   ROBERT C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A,    Stroiakovski D, et al. Improved overall survival in melanoma with    combined dabrafenib and trametinib. N Engl J Med 2015; 372:30-9.-   ROMEO Y, Zhang X, Roux P P. Regulation and function of the RSK    family of protein kinases. Biochem J 2012; 441:553-69.-   RUDOLPH J, Xiao Y, Pardi A, Ahn N G. Slow inhibition and    conformation selective properties of extracellular signal-regulated    kinase 1 and 2 inhibitors. Biochemistry 2015; 54:22-31.-   SHAUL Y D, Seger R. The MEK/ERK cascade: from signaling specificity    to diverse functions. Biochim Biophys Acta 2007; 1773:1213-26.-   SHI H, Hugo W, Kong X, Hong A, Koya R C, Moriceau G, et al. Acquired    resistance and clonal evolution in melanoma during BRAF inhibitor    therapy. Cancer Discov 2014; 4:80-93.-   SHIMA, F, Yoshikawa, Y, Ye, M, Araki, M, Matsumoto, S, Liao, J, Hu,    L, Sugimoto, T, Ijiri, Y, Takeda, A, Nishiyama, Y, Sato, C, Muraoka,    S, Tamura, A, Osoda, T, Tsuda, K-I, Miyakawa, T, Fukunishi, H,    Shimada, J, Kumasaka, Yamamoto, M, Kataoka, T. In silico discovery    of small-molecule Ras inhibitors that display antitumor activity by    blocking the Ras-effector interaction. PNAS. 2013; 110(20):8182-7.-   SCHUBBERT S, Shannon K, Bollag G. Hyperactive Ras in developmental    disorders and cancer. Nat Rev Cancer 2007; 7:295-308.-   SUN C, Hobor S, Bertotti A, Zecchin D, Huang S, Galimi F, et al.    Intrinsic resistance to MEK inhibition in KRAS mutant lung and colon    cancer through transcriptional induction of ERBB3. Cell Rep 2014;    7:86-93.-   TAFLINAR [package insert]. Research Triangle Park, N C:    GlaxoSmithKline; 2015.-   TRUNZER K, Pavlick A C, Schuchter L, Gonzalez R, McArthur G A,    Hutson T E, et al. Pharmacodynamic effects and mechanisms of    resistance to vemurafenib in patients with metastatic melanoma. J    Clin Oncol 2013; 31:1767-74.-   VILLANUEVA, J., et al. Acquired resistance to BRAF inhibitors    mediated by a RAF kinase switch in melanoma can be overcome by    cotargeting MEK and IGF-1R/PI3K. Cancer Cell. 2010; 18:683-695.-   WAGLE, N., et al. Dissecting therapeutic resistance to RAF    inhibition in melanoma by tumor genomic profiling. Journal of    Clinical Oncology 2011; 29(22):3085-3096.-   WAGLE N, Van Allen E M, Treacy D J, Frederick D T, Cooper Z A,    Taylor-Weiner A, et al. MAP kinase pathway alterations in    BRAF-mutant melanoma patients with acquired resistance to combined    RAF/MEK inhibition. Cancer Discov 2014; 4:61-8.-   Wainstein E, Seger R. The dynamic subcellular localization of ERK:    mechanisms of translocation and role in various organelles. Curr    Opin Cell Biol 2016; 39:15-20.-   WANG, H., et al. Identification of the MEK1(F129L) activating    mutation as a potential mechanism of acquired resistance to MEK    inhibition in human cancers carrying the B-RAF V600E mutation.    Cancer Res (2011); 71(16):5535-45.-   YANG W, Soares J, Greninger P, Edelman E J, Lightfoot H, Forbes S,    et al. Genomics of Drug Sensitivity in Cancer (GDSC): a resource for    therapeutic biomarker discovery in cancer cells. Nucleic Acids Res    2013; 41: D955-D961.-   YAO Z, Torres N M, Tao A, Gao Y, Luo L, Li Q, et al. BRAF Mutants    Evade ERK-Dependent Feedback by Different Mechanisms that Determine    Their Sensitivity to Pharmacologic Inhibition. Cancer Cell 2015;    28:370-83.-   YOHE S. Molecular genetic markers in acute myeloid leukemia. J Clin    Med 2015; 4:460-78.-   ZELBORAF [package insert]. South San Francisco, Calif.: Genentech    USA, Inc.; 2015.

All documents cited in this application are hereby incorporated byreference as if recited in full herein.

Although illustrative embodiments of the present invention have beendescribed herein, it should be understood that the invention is notlimited to those described, and that various other changes ormodifications may be made by one skilled in the art without departingfrom the scope or spirit of the invention.

What is claimed is:
 1. A method for treating or ameliorating the effectsof a cancer in a subject comprising: (a) identifying a subject withcancer that has become refractory or resistant to BRAF inhibitortherapy, MEK inhibitor therapy, or BRAF and MEK inhibitor therapy,comprising: (1) obtaining a biological sample from the subject; and (2)screening the sample to determine whether the subject has becomeresistant to an inhibitor therapy selected from the group consisting ofBRAF inhibitor therapy, MEK inhibitor therapy, and combinations thereof,wherein the screening for a cancer that is refractory or resistant toBRAF inhibitor therapy comprises identifying (i) a switch between RAFisoforms, (ii) upregulation of RTK signaling, (iii) reactivation ofmitogen activated protein kinase (MAPK) signaling, (iv) the presence ofa MEK activating mutation, and combinations thereof; and (b)administering to the subject with said refractory or resistant cancer aneffective amount of an ERK inhibitor, which is BVD-523 or apharmaceutically acceptable salt thereof.
 2. The method according toclaim 1, wherein the subject is a mammal.
 3. The method according toclaim 2, wherein the mammal is selected from the group consisting ofhumans, primates, farm animals, and domestic animals.
 4. The methodaccording to claim 2, wherein the mammal is a human.
 5. The methodaccording to claim 1, wherein the cancer has MAPK activity.
 6. Themethod according to claim 5, wherein the cancer is a solid tumor canceror a hematologic cancer.
 7. The method according to claim 5, wherein thecancer is selected from the group consisting of a cancer of the largeintestine, breast cancer, pancreatic cancer, skin cancer, andendometrial cancers.
 8. The method according to claim 5, wherein thecancer is melanoma.
 9. The method according to claim 1, wherein thescreening for a cancer that is refractory or resistant to MEK inhibitortherapy comprises identifying (i) amplification of mutant BRAF, (ii)STAT3 upregulation, (iii) mutations in the allosteric pocket of MEK thatdirectly block binding of inhibitors to MEK or lead to constitutive MEKactivity, and combinations thereof.
 10. The method according to claim 1further comprising administering at least one additional therapeuticagent selected from the group consisting of an antibody or fragmentthereof, a cytotoxic agent, a toxin, a radionuclide, an immunomodulator,a photoactive therapeutic agent, a radiosensitizing agent, a hormone, ananti-angiogenesis agent, and combinations thereof.
 11. The methodaccording to claim 10, wherein the additional therapeutic agent is aninhibitor of the PI3K/Akt pathway.
 12. The method according to claim 11,wherein the inhibitor of the PI3K/Akt pathway is selected from the groupconsisting of A-674563 (CAS #552325-73-2), AGL 2263, AMG-319 (Amgen,Thousand Oaks, Calif.), AS-041164(5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), AS-604850(542,2-Difluoro-benzo[1,3]dioxol-5-ylmethyleneythiazolidine-2,4-dione),AS-605240 (5-quinoxilin-6-methylene-1,3-thiazolidine-2,4-dione), AT7867(CAS #857531-00-1), benzimidazole series, Genentech (Roche HoldingsInc., South San Francisco, Calif.), BML-257 (CAS #32387-96-5), CAL-120(Gilead Sciences, Foster City, Calif.), CAL-129 (Gilead Sciences),CAL-130 (Gilead Sciences), CAL-253 (Gilead Sciences), CAL-263 (GileadSciences), CAS #612847-09-3, CAS #681281-88-9, CAS #75747-14-7, CAS#925681-41-0, CAS #98510-80-6, CCT128930 (CAS #885499-61-6), CH5132799(CAS #1007207-67-1), CHR-4432 (Chroma Therapeutics, Ltd., Abingdon, UK),FPA 124 (CAS #902779-59-3), GS-1101 (CAL-101) (Gilead Sciences), GSK690693 (CAS #937174-76-0), H-89 (CAS #127243-85-0), Honokiol, IC87114(Gilead Science), IPI-145 (Intellikine Inc.), KAR-4139 (KarusTherapeutics, Chilworth, UK), KAR-4141 (Karus Therapeutics), KIN-1(Karus Therapeutics), KT 5720 (CAS #108068-98-0), Miltefosine, MK-2206dihydrochloride (CAS #1032350-13-2), ML-9 (CAS #105637-50-1),Naltrindole Hydrochloride, OXY-111A (NormOxys Inc., Brighton, Mass.),perifosine, PHT-427 (CAS #1191951-57-1), PI3 kinase delta inhibitor,Merck KGaA (Merck & Co., Whitehouse Station, N.J.), PI3 kinase deltainhibitors, Genentech (Roche Holdings Inc.), PI3 kinase deltainhibitors, Incozen (Incozen Therapeutics, Pvt. Ltd., Hydrabad, India),PI3 kinase delta inhibitors-2, Incozen (Incozen 217 Therapeutics), PI3kinase inhibitor, Roche-4 (Roche Holdings Inc.), PI3 kinase inhibitors,Roche (Roche Holdings Inc.), PI3 kinase inhibitors, Roche-5 (RocheHoldings Inc.), PI3-alpha/delta inhibitors, Pathway Therapeutics(Pathway Therapeutics Ltd., South San Francisco, Calif.), PI3-deltainhibitors, Cellzome (Cellzome AG, Heidelberg, Germany), PI3-deltainhibitors, Intellikine (Intellikine Inc., La Jolla, Calif.), PI3-deltainhibitors, Pathway Therapeutics-1 (Pathway Therapeutics Ltd.),PI3-delta inhibitors, Pathway Therapeutics-2 (Pathway TherapeuticsLtd.), PI3-delta/gamma inhibitors, Cellzome (Cellzome AG),PI3-delta/gamma inhibitors, Cellzome (Cellzome AG), PI3-delta/gammainhibitors, Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors,Intellikine (Intellikine Inc.), PI3-delta/gamma inhibitors, PathwayTherapeutics (Pathway Therapeutics Ltd.), PI3-delta/gamma inhibitors,Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3-gamma inhibitorEvotec (Evotec), PI3-gamma inhibitor, Cellzome (Cellzome AG), PI3-gammainhibitors, Pathway Therapeutics (Pathway Therapeutics Ltd.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), PI3Kdelta/gamma inhibitors, Intellikine-1 (Intellikine Inc.), pictilisib(Roche Holdings Inc.), PIK-90 (CAS #677338-12-4), SC-103980 (Pfizer, NewYork, N.Y.), SF-1126 (Semafore Pharmaceuticals, Indianapolis, Ind.),SH-5, SH-6, Tetrahydro Curcumin, TG100-1 15 (Targegen Inc., San Diego,Calif.), Triciribine, X-339 (Xcovery, West Palm Beach, Fla.), XL-499(Evotech, Hamburg, Germany), pharmaceutically acceptable salts thereof,and combinations thereof.